Hepatitis C inhibitor tri-peptides

ABSTRACT

Racemates, diastereoisomers and optical isomers of a compound of formula (I):                    
     wherein 
     B is H, a C 6  or C 10  aryl, C 7-16  aralkyl; Het or (lower alkyl)-Het, all of which optionally substituted with C 1-6  alkyl; C 1-6  alkoxy; C 1-6  alkanoyl; hydroxy; hydroxyalkyl; halo; haloalkyl; nitro; cyano; cyanoalkyl; amino optionally substituted with C 1-6  alkyl; amido; or (lower alkyl)amide; or 
     B is an acyl derivative of formula R 4 —C(O)—; a carboxyl of formula R 4 —O—C(O)—; an amide of formula R 4 —N(R 5 )—C(O)—; a thioamide of formula R 4 —N(R 5 )—C(S)—; or a sulfonyl of formula R 4 —SO 2 ; R 5  is H or C 1-6  alkyl; and 
     Y is H or C 1-6  alkyl; 
     R 3  is C 1-8  alkyl, C 3-7  cycloalkyl, or C 4-10  alkylcycloalkyl, all optionally substituted with hydroxy, C 1-6  alkoxy, C 1-6  thioalkyl, amido, (lower alkyl)amido, C 6  or C 10  aryl, or C 7-16  aralkyl; 
     R 2  is CH 2 —R 20 , NH—R 20 , O—R 20  or S—R 20 , wherein R 20  is a saturated or unsaturated C 3-7  cycloalkyl or C 4-10  (alkylcycloalkyl), all of which being optionally mono-, di- or tri-substituted with R 21 , 
     or R 20  is a C 6  or C 10  aryl or C 7-14  aralkyl optionally substituted, or R 20  is Het or (lower alkyl)-Het, both optionally substituted, Het or (lower alkyl)-Het; carboxyl; carboxy(lower alkyl); C 6  or C 10  aryl, C 7-14  aralkyl or Het, said aryl, aralkyl or Het being optionally substituted; and 
     R 1  is H; C 1-6  alkyl, C 3-7  cycloalkyl, C 2-6  alkenyl, or C 2   6  alkynyl, all optionally substituted with halogen; or a pharmaceutically acceptable salt or ester thereof.

This application is a divisional of U.S. application Ser. No.09/368,866, filed on Aug. 5, 1999, U.S. Pat. No. 6,323,180 which claimsthe benefit of U.S. Provisional Application No. 60/095,931, filed Aug.10, 1998, and U.S. Provisional Application No. 60/132,386, filed May 4,1999.

FIELD OF THE INVENTION

The present invention relates to compounds, process for their synthesis,compositions and methods for the treatment of hepatitis C virus (HCV)infection. In particular, the present invention provides novel peptideanalogs, pharmaceutical compositions containing such analogs and methodsfor using these analogs in the treatment of HCV infection. The presentinvention also provides processes and intermediates for the synthesis ofthese peptide analogs.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is the major etiological agent ofpost-transfusion and community-acquired non-A non-B hepatitis worldwide.It is estimated that over 150 million people worldwide are infected bythe virus. A high percentage of carriers become chronically infected andmany progress to chronic liver disease, so-called chronic hepatitis C.This group is in turn at high risk for serious liver disease such asliver cirrhosis, hepatocellular carcinoma and terminal liver diseaseleading to death.

The mechanism by which HCV establishes viral persistence and causes ahigh rate of chronic liver disease has not been thoroughly elucidated.It is not known how HCV interacts with and evades the host immunesystem. In addition, the roles of cellular and humoral immune responsesin protection against HCV infection and disease have yet to beestablished. Immunoglobulins have been reported for prophylaxis oftransfusion-associated viral hepatitis, however, the Center for DiseaseControl does not presently recommend immunoglobulins treatment for thispurpose. The lack of an effective protective immune response ishampering the development of a vaccine or adequate post-exposureprophylaxis measures, so in the near-term, hopes are firmly pinned onantiviral interventions.

Various clinical studies have been conducted with the goal ofidentifying pharmaceutical agents capable of effectively treating HCVinfection in patients afflicted with chronic hepatitis C. These studieshave involved the use of interferon-alpha, alone and in combination withother antiviral agents. Such studies have shown that a substantialnumber of the participants do not respond to these therapies, and ofthose that do respond favorably, a large proportion were found torelapse after termination of treatment.

Until recently, interferon (IFN) was the only available therapy ofproven benefit approved in the clinic for patients with chronichepatitis C. However the sustained response rate is low, and interferontreatment also induces severe side-effects (i.e. retinopathy,thyroiditis, acute pancreatitis, depression) that diminish the qualityof life of treated patients. Recently, interferon in combination withribavirin has been approved for patients non-responsive to IFN alone.However, the side effects caused by IFN are not alleviated with thiscombination therapy. Therefore, a need exists for the development ofeffective antiviral agents for treatment of HCV infection that overcomesthe limitations of existing pharmaceutical therapies.

HCV is an enveloped positive strand RNA virus in the Flaviviridaefamily. The single strand HCV RNA genome is approximately 9500nucleotides in length and has a single open reading frame (ORF) encodinga single large polyprotein of about 3000 amino acids. In infected cells,this polyprotein is cleaved at multiple sites by cellular and viralproteases to produce the structural and non-structural (NS) proteins. Inthe case of HCV, the generation of mature nonstructural proteins (NS2,NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. Thefirst one, as yet poorly characterized, cleaves at the NS2-NS3 junction;the second one is a serine protease contained within the N-terminalregion of NS3 (henceforth referred to as NS3 protease) and mediates allthe subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4Acleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A,NS5A-NS5B sites. The NS4A protein appears to serve multiple functions,acting as a cofactor for the NS3 protease and possibly assisting in themembrane localization of NS3 and other viral replicase components. Thecomplex formation of the NS3 protein with NS4A seems necessary to theprocessing events, enhancing the proteolytic efficiency at all of thesites. The NS3 protein also exhibits nucleoside triphosphatase and RNAhelicase activities. NS5B is a RNA-dependent RNA polymerase that isinvolved in the replication of HCV. A general strategy for thedevelopment of antiviral agents is to inactivate virally encoded enzymesthat are essential for the replication of the virus. In this vein,patent application WO 97/06804 describes the (−) enantiomer of thenucleoside analogue cytosine-1,3-oxathiolane (also known as 3TC) asactive against HCV. This compound, although reported as safe in previousclinical trials against HIV and HBV, has yet to be clinically provenactive against HCV and its mechanism of action against the virus has yetto be reported.

Intense efforts to discover compounds which inhibit the NS3 protease orRNA helicase of HCV have led to the following disclosures:

U.S. Pat. No. 5,633,388 describes heterocyclic-substituted carboxamidesand analogues as being active against HCV. These compounds are directedagainst the helicase activity of the NS3 protein of the virus butclinical tests have not yet been reported.

A phenanthrenequinone has been reported by Chu et al., (Tet. Lett.,(1996), 7229-7232) to have activity against the HCV NS3 protease invitro. No further development on this compound has been reported.

A paper presented at the Ninth International Conference on AntiviralResearch, Urabandai, Fukyshima, Japan (1996) (Antiviral Research,(1996), 30, 1, A23 (abstract 19)) reports thiazolidine derivatives to beinhibitory to the HCV protease.

Several studies have reported compounds inhibitory to other serineproteases, such as human leukocyte elastase. One family of thesecompounds is reported in WO 95/33764 (Hoechst Marion Roussel, 1995). Thepeptides disclosed in this application aremorpholinylcarbonyl-benzoyl-peptide analogues that are structurallydifferent from the peptides of the present invention.

WO 98/17679 from Vertex Pharmaceuticals Inc. discloses inhibitors ofserine protease, particularly, Hepatitis C virus NS3 protease. Theseinhibitors are peptide analogues based on the NS5A/5B natural substrate.Although several tripeptides are disclosed, all of these peptideanalogues contain C-terminal activated carbonyl function as an essentialfeature. These analogues were also reported to be active against otherserine protease and are therefore not specific for HCV NS3 protease.

Hoffman LaRoche has also reported hexapeptides that are proteinaseinhibitors useful as antiviral agents for the treatment of HCVinfection. These peptides contain an aldehyde or a boronic acid at theC-terminus.

Steinkühler et al. and Ingallinella et al. have published on NS4A4Bproduct inhibition (Biochemistry (1998), 37, 8899-8905 and 8906-8914).However, the peptides and peptide analogues presented do not include nordo they lead to the design of the peptides of the present invention.

One advantage of the present invention is that it provides tripeptidesthat are inhibitory to the NS3 protease of the hepatitis C virus.

A further advantage of one aspect of the present invention resides inthe fact that these peptides specifically inhibit the NS3 protease anddo not show significant inhibitory activity at concentrations up to 300μM against other serine proteases such as human leukocyte elastase(HLE), porcine pancreatic elastase (PPE), or bovine pancreaticchymotrypsin, or cysteine proteases such as human liver cathepsin B (CatB).

A further advantage of the present invention is that it provides smallpeptides of low molecular weight that may be capable of penetrating cellmembranes and may be active in cell culture and in vivo with goodpharmacokinetic profile.

SUMMARY OF THE INVENTION

Included in the scope of the invention are racemates, diastereoisomersand optical isomers of a compound of formula (I):

wherein

B is H, a C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl; Het or (lower alkyl)-Het, allof which optionally substituted with C₁₋₆ alkyl; C₁₋₆ alkoxy; C₁₋₆alkanoyl; hydroxy; hydroxyalkyl; halo; haloalkyl; nitro; cyano;cyanoalkyl; amino optionally substituted with C₁₋₆ alkyl; amido; or(lower alkyl)amide;

or B is an acyl derivative of formula R₄—C(O)—; a carboxyl of formulaR₄—O—C(O)—; an amide of formula R₄—N(R₅)—C(O)—; a thioamide of formulaR₄—N(R₅)—C(S)—; or a sulfonyl of formula R₄—SO₂ wherein

R₄ is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido, or (lower alkyl) amide;

(ii) C₃₋₇ cycloalkyl, C₃₋₇ cycloalkoxy, or C₄₋₁₀ alkylcycloalkyl, alloptionally substituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl,amino optionally mono- or di-substituted with C₁₋₆ alkyl, amido, or(lower alkyl) amide;

(iii) amino optionally mono- or di-substituted with C-₁₋₆ alkyl; amido;or (lower alkyl)amide;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionallymono- or di-substituted with C₁₋₆ alkyl; or

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amido, (lower alkyl) amide, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl;

R₅ is H or C₁₋₆ alkyl;

with the proviso that when R₄ is an amide or a thioamide, R₄ is not (ii)a cycloalkoxy; and

Y is H or C₁₋₆ alkyl;

R³ is C₁₋₈ alkyl, C₃₋₇ cycloalkyl, or C₄₋₁₀ alkylcycloalkyl, alloptionally substituted with hydroxy, C₁₋₆ alkoxy, C-₁₋₆ thioalkyl,amido, (lower alkyl)amido, C₆ or C₁₀ aryl, or C₇₋₁₆ aralkyl;

R² is CH₂—R₂₀, NH—R₂₀, O—R₂₀ or S—R₂₀, wherein R₂₀ is a saturated orunsaturated C₃₋₇ cycloalkyl or C₄₋₁₀ (alkylcycloalkyl), all of whichbeing optionally mono-, di- or tri-substituted with R₂₁,

or R₂₀ is a C₆ or C₁₀ aryl or C₇₋₁₄ aralkyl, all optionally mono-, di-or tri-substituted with R₂₁,

or R₂₀ is Het or (lower alkyl)-Het, both optionally mono-, di- ortri-substituted with R₂₁,

wherein each R₂₁ is independently C₁₋₆ alkyl; C₁₋₆ alkoxy; lowerthioalkyl;

sulfonyl; NO₂; OH; SH; halo; haloalkyl; amino optionally mono- ordi-substituted with C₁₋₆ alkyl, C₆ or C₁₀ aryl, C₇₋₁₄ aralkyl, Het or(lower alkyl)-Het;

amido optionally mono-substituted with C₁₋₆ alkyl, C₆ or C₁₀ aryl, C₇₋₁₄aralkyl, Het or (lower alkyl)-Het;

carboxyl; carboxy(lower alkyl); C₆ or C₁₀ aryl, C₇₋₁₄ aralkyl or Het,said aryl, alkyl or Het being optionally substituted with R₂₂;

wherein R₂₂ is C₁₋₆ alkyl; C₃₋₇ cycloalkyl; C₁₋₆ alkoxy; aminooptionally mono- or di-substituted with C₁₋₆ alkyl; sulfonyl; (loweralkyl)sulfonyl; NO₂; OH; SH; halo; haloalkyl; carboxyl; amide; (loweralkyl)amide; or Het optionally substituted with C₁₋₆ alkyl

R¹ is H, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl, alloptionally substituted with halogen;

or a pharmaceutically acceptable salt or ester thereof.

Included within the scope of this invention is a pharmaceuticalcomposition comprising an anti-hepatitis C virally effective amount of acompound of formula I, or a therapeutically acceptable salt or esterthereof, in admixture with a pharmaceutically acceptable carrier mediumor auxiliary agent.

An important aspect of the invention involves a method of treating ahepatitis C viral infection in a mammal by administering to the mammalan anti-hepatitis C virally effective amount of the compound of formulaI,or a therapeutically acceptable salt or ester thereof or a compositionas described above.

Another important aspect involves a method of inhibiting the replicationof hepatitis C virus by exposing the virus to a hepatitis C viral NS3protease inhibiting amount of the compound of formula I, or atherapeutically acceptable salt or ester thereof or a composition asdescribed above.

Still another aspect involves a method of treating a hepatitis C viralinfection in a mammal by administering thereto an anti-hepatitis Cvirally effective amount of a combination of the compound of formula I,or a therapeutically acceptable salt or ester thereof. According to oneembodiment, the pharmaceutical compositions of this invention comprisean additional immunomodulatory agent. Examples of additionalimmunomodulatory agents include but are not limited to, α-, β-, andδ-interferons.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

As used herein, the following definitions apply unless otherwise noted:With reference to the instances where (R) or (S) is used to designatethe configuration of a substituent, e.g. R¹ of the compound of formulaI, the designation is done in the context of the compound and not in thecontext of the substituent alone.

The natural amino acids, with exception of glycine, contain a chiralcarbon atom. Unless otherwise specifically indicated, the compoundscontaining natural amino acids with the L-configuration are preferred.However, applicants contemplate that when specified, some amino acids ofthe formula I can be of either D- or L-configuration or can be mixturesof D- and L-isomers, including racemic mixtures. The designation “P1, P2and P3” as used herein refer to the position of the amino acid residuesstarting from the C-terminus end of the peptide analogues and extendingtowards the N-terminus [i.e. P1 refers to position 1 from theC-terminus, P2: second position from the C-terminus, etc.) (see BergerA. & Schechter I., Transactions of the Royal Society London series(1970), B257, 249-264]. The abbreviations for the α-amino acids used inthis application are set forth in Table A.

TABLE A Amino Acid Symbol 1-aminocyclopropyl-carboxylic acid AccaAlanine Ala Aspartic acid Asp Cysteine Cys Cyclohexylglycine (alsonamed: 2-amino-2- Chg cyclohexylacetic acid) Glutamic acid GluIsoleucine Ile Leucine Leu Phenylalanine Phe Proline Pro Valine Valtert-Butylglycine Tbg

As used herein the term “1-aminocyclopropyl-carboxylic acid” (Acca)refers to a compound of formula:

As used herein the term “tert-butylglycine” refers to a compound offormula:

The term “residue” with reference to an amino acid or amino acidderivative means a radical derived from the corresponding α-amino acidby eliminating the hydroxyl of the carboxy group and one hydrogen of theα-amino group. For instance, the terms Gln, Ala, Gly, IIe, Arg, Asp,Phe, Ser, Leu, Cys, Asn, Sar and Tyr represent the “residues” ofL-glutamine, L-alanine, glycine, L-isoleucine, L-arginine, L-asparticacid, L-phenylalanine, L-serine, L-leucine, L-cysteine, L-asparagine,sarcosine and L-tyrosine, respectively.

The term “side chain” with reference to an amino acid or amino acidresidue means a group attached to the α-carbon atom of the a-amino acid.For example, the R-group side chain for glycine is hydrogen, for alanineit is methyl, for valine it is isopropyl. For the specific R-groups orside chains of the α-amino acids reference is made to A. L. Lehninger'stext on Biochemistry (see chapter 4).

The term “halo” as used herein means a halogen substituent selected frombromo, chloro, fluoro or iodo.

The term “C₁₋₆ alkyl” or “(lower)alkyl” as used herein, either alone orin combination with another substituent, means acyclic, straight orbranched chain alkyl substituents containing from 1 to six carbon atomsand includes, for example, methyl, ethyl, propyl, butyl, tert-butyl,hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.

The term “C₃₋₇ cycloalkyl” as used herein, either alone or incombination with another substituent, means a cycloalkyl substituentcontaining from three to seven carbon atoms and includes cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. This term alsoincludes “spiro”-cyclic group such as spiro-cyclopropyl orspiro-cyclobutyl:

The term “unsaturated cycloalkyl” includes, for example, cyclohexenyl:

The term “C₄₋₁₀ (alkylcycloalkyl) as used herein means a cycloalkylradical containing from three to seven carbon atoms linked to an alkylradical, the linked radicals containing up to ten carbon atoms; forexample, cyclopropylmethyl, cyclopentylethyl, cyclohexylmethyl,cyclohexylethyl or cycloheptylethyl.

The term “C₂₋₁₀ alkenyl” as used herein, either alone or in combinationwith another radical, means an alkyl radical as defined above containingfrom 2 to 10 carbon atoms, and further containing at least one doublebond. For example alkenyl includes allyl and vinyl.

The term “C₁₋₆ alkanoyl” as used herein, either alone or in combinationwith another radical, means straight or branched 1-oxoalkyl radicalscontaining one to six carbon atoms and includes formyl, acetyl,1-oxopropyl (propionyl), 2-methyl-1-oxopropyl, 1-oxohexyl and the like.

The term “C₁₋₆ alkoxy” as used herein, either alone or in combinationwith another radical, means the radical —O(C₁₋₆alkyl) wherein alkyl isas defined above containing up to six carbon atoms. Alkoxy includesmethoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy.The latter radical is known commonly as tert-butoxy. The term “C₃₋₇cycloalkoxy” as used herein, either alone or in combination with anotherradical, means a C₃₋₇ cycloalkyl group linked to an oxygen atom, suchas, for example:

The term “C₆ or C₁₀ aryl” as used herein, either alone or in combinationwith another radical, means either an aromatic monocyclic groupcontaining 6 carbon atoms or an aromatic bicyclic group containing 10carbon atoms. For example, aryl includes phenyl, 1-naphthyl or2-naphthyl.

The term “C₇₋₁₆ aralkyl” as used herein, either alone or in combinationwith another radical, means a C₆ or C₁₀ aryl as defined above linked toan alkyl group, wherein alkyl is as defined above containing from 1 to 6carbon atoms. C₇₋₁₆ aralkyl includes for example benzyl, butylphenyl,and 1-naphthylmethyl.

The term “amino aralkyl” as used herein, either alone or in combinationwith another radical, means an amino group substituted with a C₇₋₁₆aralkyl group, such as, for example, the amino aralkyl:

The term “(lower alkyl)amide” as used herein, either alone or incombination with another radical, means an amide mono-substituted with aC₁₋₆ alkyl, such as:

The term “carboxy(lower)alkyl” as used herein, either alone or incombination with another radical, means a carboxyl group (COOH) linkedthrough a (lower)alkyl group as defined above and includes for examplebutyric acid.

The term “heterocycle” or “Het” as used herein, either alone or incombination with another radical, means a monovalent radical derived byremoval of a hydrogen from a five-, six-, or seven-membered saturated orunsaturated (including aromatic) heterocycle containing from one to fourheteroatoms selected from nitrogen, oxygen and sulfur. Furthermore,“Het” as used herein, means a heterocycle as defined above fused to oneor more other cycle, be it a heterocycle or any other cycle. Examples ofsuitable heterocycles include: pyrrolidine, tetrahydrofuran,thiazolidine, pyrrole, thiophene, diazepine, 1H-imidazole, isoxazole,thiazole, tetrazole, piperidine, 1,4-dioxane, 4-morpholine, pyridine,pyrimidine, thiazolo[4,5-b]-pyridine, quinoline, or indole, or thefollowing heterocycles:

The term “(lower alkyl)-Het” as used herein, means a heterocyclicradical as defined above linked through a chain or branched alkyl group,wherein alkyl is as defined above containing from 1 to 6 carbon atoms.Examples of (lower alkyl)-Het include:

The term “pharmaceutically acceptable ester” as used herein, eitheralone or in combination with another substituent, means esters of thecompound of formula I in which any of the carboxyl functions of themolecule, but preferably the carboxy terminus, is replaced by analkoxycarbonyl function:

in which the R moiety of the ester is selected from alkyl (e.g. methyl,ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g. methoxymethyl);alkoxyacyl (e.g. acetoxymethyl); aralkyl (e.g. benzyl); aryloxyalkyl(e.g. phenoxymethyl); aryl (e.g. phenyl), optionally substituted withhalogen, C₁₋₄ alkyl or C₁₋₄ alkoxy. Other suitable prodrug esters can befound in Design of prodrugs, Bundgaard, H. Ed. Elsevier (1985)incorporated herewith by reference. Such pharmaceutically acceptableesters are usually hydrolyzed in vivo when injected in a mammal andtransformed into the acid form of the compound of formula I.

With regard to the esters described above, unless otherwise specified,any alkyl moiety present advantageously contains 1 to 16 carbon atoms,particularly 1 to 6 carbon atoms. Any aryl moiety present in such estersadvantageously comprises a phenyl group.

In particular the esters may be a C₁₋₆ alkyl ester, an unsubstitutedbenzyl ester or a benzyl ester substituted with at least one halogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, nitro or trifluoromethyl.

The term “pharmaceutically acceptable salt” as used herein includesthose derived from pharmaceutically acceptable bases. Examples ofsuitable bases include choline, ethanolamine and ethylenediamine. Na⁺,K⁺, and Ca⁺⁺salts are also contemplated to be within the scope of theinvention (also see Pharmaceutical salts, Birge, S. M. et al., J. Pharm.Sci., (1977), 66, 1-19, incorporated herein by reference).

Preferred Embodiments

Included within the scope of this invention are compounds of formula Iwherein Preferably, B is a C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, alloptionally substituted with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl,hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido,(lower alkyl)amido, or amino optionally substituted with C₁₋₆ alkyl; or

B is preferably Het or (lower alkyl)-Het, all optionally substitutedwith C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl, hydroxy, hydroxyalkyl,halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, oramino optionally substituted with C₁₋₆ alkyl.

Alternatively, B is preferably R₄—SO₂ wherein R₄ is preferably C₁₋₆alkyl; amido; (lower alkyl)amide; C₆ or C₁₀ aryl, C₇₋₁₄ aralkyl or Het,all optionally substituted with C₁₋₆ alkyl.

Alternatively, B is preferably an acyl derivative of formula R₄—C(O)—wherein R₄ is preferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, hydroxy or C₁₋₆alkoxy, amido, (lower alkyl)amide, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, both optionallysubstituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, or amino optionally mono- or di-substituted with C₁₋₆alkyl;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionallysubstituted with C₁₋₆ alkyl;

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amino optionally substituted with C₁₋₆ alkyl, amido,(lower alkyl)amide, or amino optionally substituted with C₁₋₆ alkyl.

Alternatively, B is preferably a carboxyl of formula R₄—O—C(O)—, whereinR₄ is preferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido or (lower alkyl)amide;

(ii) C₃₋₇ cycloalkyl, C₄₋₁₀ alkylcycloalkyl, all optionally substitutedwith carboxyl, (C₁₋₆ alkoxy)carbonyl, amino optionally mono- ordi-substituted with C₁₋₆ alkyl, amido or (lower alkyl)amide;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl optionally substituted with C₁₋₆alkyl, hydroxy, amido, (lower alkyl)amido, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl; or

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido or (lower alkyl)amido.

Alternatively, B is preferably an amide of formula R₄—N(R₅)—C(O)—wherein R₄ is preferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amido, (lower alkyl)amido, or amino optionallymono- or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amido, or amino optionally mono- or di-substituted with C₁₋₆alkyl;

(iii) amino optionally mono- or di-substituted with C₁₋₃ alkyl;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionallysubstituted with C₁₋₆ alkyl; or

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amino optionally substituted with C₁₋₆ alkyl, amido or(lower alkyl)amide; and

R₅ is preferably H or methyl.

Alternatively, B is a preferably thioamide of formula R₄—NH—C(S)—;wherein R₄ is preferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl orC₁₋₆ alkoxy;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amino or amido.

More preferably, B is a C₆ or C₁₀ aryl optionally substituted with C₁₋₆alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl, hydroxy, hydroxyalkyl, halo,haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amide, or aminooptionally mono- or di-substituted with C₁₋₆ alkyl, such that B is forexample:

or B is more preferably Het optionally substituted with C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ alkanoyl, hydroxy, halo, amido, (lower alkyl)amide, oramino optionally mono- or di-substituted with C₁₋₆ alkyl, such that B isfor example:

Alternatively, B is more preferably R₄—SO₂ wherein R₄ is preferably C₆or C₁₀ aryl, a C₇₋₁₄ aralkyl or Het all optionally substituted with C₁₋₆alkyl; amido, (lower alkyl)amide, such that B is, for example:

Alternatively, B is more preferably an acyl derivative of formulaR₄—C(O)— wherein R₄ is preferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, hydroxy or C₁₋₆alkoxy; or

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, both optionallysubstituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, such that Bis, for example:

or R₄ is preferably

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, such that B is for example:

or R₄ is preferably

(v) Het optionally substituted with C₁₋₆ alkyl, hydroxy, amido or amino,such that B is for example:

Alternatively, B is more preferably a carboxyl of formula R₄—O—C(O)—,wherein R₄ is preferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy or amido, (lower alkyl)amide, amino optionallymono- or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl, C₄₋₁₀ alkylcycloalkyl, all optionally substitutedwith carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (lower alkyl)amide, aminooptionally mono- or di-substituted with C₁₋₆ alkyl, such that B is forexample:

or R₄ is preferably

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amino optionally substituted with C₁₋₆ alkyl; or

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amido, or amino optionally mono-substituted with C₁₋₆alkyl, such that B is for example:

Alternatively, B is more preferably an amide of formula R₄—N(R₅)—C(O)—wherein R₄ is preferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amido, (lower alkyl)amide, amino optionally mono-or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, amino optionally mono- or di-substituted with C₁₋₆ alkyl;and

R₅ is H or methyl, such that B is for example:

or R₄ is preferably

(iii) amino optionally mono- or di-substituted with C₁₋₃ alkyl, suchthat B is for example:

or R₄ is preferably

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amino or amido optionally substituted with C₁₋₆alkyl; or

(v) Het optionally substituted with C₁₋₆ alkyl, hydroxy, amino or amido,such that B is for example:

Alternatively, B is more preferably a thioamide of formula R₄—NH—C(S)—;wherein R₄ is preferably

R₄ is (i) C₁₋₁₀ alkyl; or (ii) C₃₋₇ cycloalkyl, such that B is forexample:

Most preferably, B is an amide of formula R₄—NH—C(O)— wherein R₄ ispreferably

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy amido, (lower alkyl)amide, amino optionally mono-or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, amino optionally mono- or di-substituted with C₁₋₆ alkyl;

or R₄ is preferably

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl optionally substituted with C₁₋₆alkyl, hydroxy, amino or amido, such that B is for example:

Even most preferably, B is tert-butoxycarbonyl (Boc) or

Preferably, Y is H or methyl. More preferably, Y is H.

Preferably, R³ is C₁₋₈ alkyl, C₃₋₇ cycloalkyl, or C₄₋₁₀ alkylcycloalkyl,all optionally substituted with hydroxy, C₁₋₆ alkoxy, C₁₋₆ thioalkyl,acetamido, C₆ or C₁₀ aryl, or C₇₋₁₆ aralkyl, such that B is for example:

More preferably, R³ is the side chain of tert-butylglycine (Tbg), IIe,Val, Chg or:

Most preferably, R³ is the side chain of Tbg, Chg or Val.

Included within the scope of the invention are compounds of formula Iwherein, preferably, R² is S—R₂₀ or O—R₂₀ wherein R₂₀ is preferably a C₆or C₁₀ aryl, C₇₋₁₆ aralkyl, Het or —CH₂—Het, all optionally mono-, di-or tri-substituted with R₂₁.

Preferably, R₂₁ is C₁₋₆ alkyl; C₁₋₆ alkoxy; lower thioalkyl; amino oramido optionally mono-or di-substituted with C₁₋₆ alkyl, C₆ or C₁₀ aryl,C₇₋₁₆ aralkyl, Het or (lower alkyl)-Het; NO₂; OH; halo; trifluoromethyl;carboxyl; C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl, or Het, said aryl, aralkyl orHet being optionally substituted with R₂₂. More preferably, R₂₁ is C₁₋₆alkyl; C₁₋₆alkoxy; amino; di(lower alkyl)amino; (lower alkyl)amide; C₆or C₁₀ aryl, or Het, said aryl or Het being optionally substituted withR₂₂.

Preferably, R₂₂ is C₁₋₆alkyl; C₃₋₇ cycloalkyl; C₁₋₆alkoxy; amino; mono-or di-(lower alkyl)amino; (lower alkyl)amide; sulfonylalkyl; NO₂; OH;halo; trifluoromethyl; carboxyl or Het. More preferably, R₂₂ is C₁₋₆alkyl; C₃₋₇ cycloalkyl; C₁₋₆ alkoxy; amino; mono- or di(loweralkyl)amino; amido; (lower alkyl)amide; halo; trifluoromethyl or Het.

Most preferably, R₂₂ is C₁₋₆ alkyl; C₁₋₆ alkoxy; halo; amino optionallymono- or di-substituted with lower alkyl; amido; (lower alkyl)amide; orHet. Even most preferably, R₂₂ is methyl; ethyl; isopropyl; tert-butyl;methoxy; chloro; amino optionally mono- or di-substituted with loweralkyl; amido, (lower alkyl)amide; or (lower alkyl) 2-thiazole.

Alternatively, R² is preferably selected from the group consisting of:

More preferably, R² is 1-naphthylmethoxy; 2-naphthylmethoxy; benzyloxy,1-naphthyloxy; 2-naphthyloxy; or quinolinoxy unsubstituted, mono- ordi-substituted with R₂₁ as defined above. Most preferably, R² is1-naphtylmethoxy; or quinolinoxy unsubstituted, mono- or di-substitutedwith R₂₁ as defined above, such that R² is for example:

Still, more preferably, R² is

More preferably, R_(21A) is C₁₋₆ alkyl such as isopropyl, tert-butyl orcyclohexyl;

C₁₋₆ alkoxy such as methoxy,

lower thioalkyl such as

halo such as chloro;

amino optionally mono-substituted with C₁₋₆ alkyl; or C₆ or C₁₀ aryl,such that R_(21A) is for example: dimethylamino, Ph—N(Me)—;unsubstituted C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl, such as for example phenylor

or R_(21A) is more preferably Het optionally substituted with R₂₂wherein R₂₂ is C₁₋₆ alkyl, C₁₋₆ alkoxy, amido, (lower alkyl)amide, aminooptionally mono- or di-substituted with C₁₋₆ alkyl, or Het, such thatR_(21A) is for example:

Most preferably, R_(21A) is C₆, C₁₀ aryl or Het, all optionallysubstituted with R₂₂ as defined above, such that R_(21A) is for example:

Even most preferably, R² is:

wherein R_(22A) is preferably C₁₋₆ alkyl (such as methyl); C₁₋₆ alkoxy(such as methoxy); or halo (such as chloro); R_(22B) is preferably C₁₋₆alkyl, amino optionally mono-substituted with C₁₋₆ alkyl, amido, or(lower alkyl)amide; and R_(21B) is preferably C₁₋₆ alkyl, C₁₋₆ alkoxy,amino, di(lower alkyl)amino, (lower alkyl)amide, NO₂, OH, halo,trifluoromethyl, or carboxyl. More preferably, R_(21B) is C₁₋₆ alkoxy,or di(lower alkyl)amino. Most preferably, R_(21B) is methoxy.

As described hereinabove the P1 segment of the compounds of formula I isa cyclobutyl or cyclopropyl ring, both optionally substituted with R¹.

Preferably, R¹ is H, C₁₋₃ alkyl, C₃₋₅ cycloalkyl, or C₂₋₄ alkenyloptionally substituted with halo. More preferably R¹ is ethyl, vinyl,cyclopropyl, 1 or 2-bromoethyl or 1 or 2-bromovinyl. Most preferably, R¹is vinyl.

When R¹ is not H, then P1 is preferably a cyclopropyl system of formula:

wherein C₁ and C₂ each represent an asymmetric carbon atom at positions1 and 2 of the cyclopropyl ring. Notwithstanding other possibleasymmetric centers at other segments of the compounds of formula I thepresence of these two asymmetric centers means that the compounds offormula I can exist as racemic mixtures of diastereoisomers. Asillustrated in the examples hereinafter, the racemic mixtures can beprepared and thereafter separated into individual optical isomers, orthese optical isomers can be prepared by chiral synthesis.

Hence, the compound of formula I can exist as a racemic mixture ofdiastereoisomers at carbon 1 but wherein R¹ at carbon 2 is orientatedsyn to the carbonyl at position 1, represented by the radical:

or the compound of formula I can exist as a racemic mixture ofdiastereoisomers wherein R¹ at position 2 is orientated anti to thecarbonyl at position 1, represented by the radical:

In turn, the racemic mixtures can be separated into individual opticalisomers. A most interesting finding of this invention pertains to theaddition of a R¹ substituent on the carbon 2 as well as the spatialorientation of the P1 segment. The finding concerns the configuration ofthe asymmetric carbon 1. A preferred embodiment is one wherein R¹ is notH and carbon 1 has the R configuration.

More explicitly, the introduction of a substituent (R¹) at C2 has animpact on the potency when R¹ is introduced in a way that C1 has the Rconfiguration. For example compounds 901 (1R,2S) and 203 (1R,2R) haveactivities of 25 and 82 nM respectively. When compared to theunsubstituted cyclopropyl compound 111 (475 nM), a substantial increasein potency is observed. Moreover, as shown for compounds 901 and 203when carbon 1 has the R configuration, HCV NS3 protease inhibition isfurther enhanced by the configuration of the substituent R¹ (e.g. alkylor alkylene) at carbon 2 of the cyclopropyl ring, e.g. the compound thatpossesses R¹ “syn” to the carboxyl has greater potency (25 nM) than the“anti” enantiomer (82 nM). We can see the effect of the R vs. Sconfiguration at C1 by comparing compounds 801(1R,2S) and itscorresponding (1S,2S) isomer which have potencies of 6 nM and >10 μMrespectively, a difference of over 1500 fold!! Therefore a mostpreferred compound is an optical isomer having the R¹ substituent andthe carbonyl in a syn orientation in the following absoluteconfiguration:

In the case where R¹ is ethyl, for example, the asymmetric carbon atomsat positions 1 and 2 have the R,R configuration.

Included within the scope of this invention are compounds of formula Iwherein

B is a C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl, hydroxy, hydroxyalkyl, halo,haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or aminooptionally substituted with C₁₋₆ alkyl; or Het or (lower alkyl)-Het, alloptionally substituted with C₁₋₆ alkyl, C ₁₋₆ alkoxy, C₁₋₆ alkanoyl,hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido,(lower alkyl)amido, or amino optionally substituted with C₁₋₆ alkyl, or

B is R₄—SO₂ wherein R₄ is preferably amido; (lower alkyl)amide; C₆ orC₁₀ aryl, C₇₋₁₄ aralkyl or Het, all optionally substituted with C₁₋₆alkyl, or

B is an acyl derivative of formula R₄—C(O)— wherein R₄ is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, hydroxy or C₁₋₆alkoxy, amido, (lower alkyl)amide, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, both optionallysubstituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, or amino optionally mono- or di-substituted with C₁₋₆alkyl;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionallysubstituted with C₁₋₆ alkyl;

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amino optionally substituted with C₁₋₆ alkyl, amido,(lower alkyl)amide, or amino optionally substituted with C₁₋₆ alkyl, or

B is a carboxyl of formula R₄—O—C(O)—, wherein R₄ is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido or (lower alkyl)amide;

(ii) C₃₋₇ cycloalkyl, C₄₋₁₀ alkylcycloalkyl, all optionally substitutedwith carboxyl, (C₁₋₆ alkoxy)carbonyl, amino optionally mono- ordi-substituted with C₁₋₆ alkyl, amido or (lower alkyl)amide;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl optionally substituted with C₁₋₆alkyl, hydroxy, amido, (lower alkyl)amido, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl; or

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido or (lower alkyl)amido, or

B is an amide of formula R₄—N(R₅)—C(O)— wherein R₄ is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amido, (lower alkyl)amido, or amino optionallymono- or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amido, or amino optionally mono- or di-substituted with C₁₋₆alkyl;

(iii) amino optionally mono- or di-substituted with C₁₋₃ alkyl;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionallysubstituted with C₁₋₆ alkyl; or

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amino optionally substituted with C₁₋₆ alkyl, amido or(lower alkyl)amide; and

R₅ is preferably H or methyl, or

B is thioamide of formula R₄—NH—C(S)—; wherein R₄ is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl orC₁₋₆ alkoxy;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amino or amido;

Y is H or methyl;

R³ is C₁₋₈ alkyl, C₃₋₇ cycloalkyl, or C₄₋₁₀ alkylcycloalkyl, alloptionally substituted with hydroxy, C₁₋₆ alkoxy, C₁₋₆ thioalkyl,acetamido, C₆ or C₁₀ aryl, or C₇₋₁₆ aralkyl;

R² is S—R₂₀ or O—R₂₀ wherein R₂₀ is preferably a C₆ or C₁₀ aryl, C₇₋₁₆aralkyl, Het or —CH₂—Het, all optionally mono-, di- or tri-substitutedwith R₂₁, wherein

R₂₁ is C₁₋₆ alkyl; C₁₋₆ alkoxy; lower thioalkyl; amino or amidooptionally mono- or di-substituted with C₁₋₆ alkyl, C₆ or C₁₀ aryl,C₇₋₁₆ aralkyl, Het or (lower alkyl)-Het; NO₂; OH; halo; trifluoromethyl;carboxyl; C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl, or Het, said aryl, aralkyl orHet being optionally substituted with R₂₂, wherein

R₂₂ is C₁₋₆ alkyl; C₃₋₇ cycloalkyl; C₁₋₆ alkoxy; amino; mono- ordi-(lower alkyl)amino; (lower alkyl)amide; sulfonylalkyl; NO₂; OH; halo;trifluoromethyl; carboxyl or Het; or

R² is selected from the group consisting of:

or R² is 1-naphthylmethoxy; 2-naphthylmethoxy; benzyloxy, 1-naphthyloxy;2-naphthyloxy; or quinolinoxy unsubstituted, mono- or di-substitutedwith R₂ as defined above;

the P1 segment is a cyclobutyl or cyclopropyl ring, both optionallysubstituted with R¹, wherein R¹ is H, C₁₋₃ alkyl, C₃₋₅ cycloalkyl, orC₂₋₄ alkenyl optionally substituted with halo, and said R¹ at carbon 2is orientated syn to the carbonyl at position 1, represented by theradical:

Included within the scope of this invention are compounds of formula Iwherein B is a C₆ or C₁₀ aryl optionally substituted with C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl,nitro, cyano, cyanoalkyl, amido, (lower alkyl)amide, or amino optionallymono- or di-substituted with C₁₋₆ alkyl; or B is Het optionallysubstituted with C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆ alkanoyl, hydroxy, halo,amido, (lower alkyl)amide, or amino optionally mono- or di-substitutedwith C₁₋₆ alkyl; or B is R₄—SO₂ wherein R₄ is C₆ or C₁₀ aryl, a C₇₋₁₄aralkyl or Het all optionally substituted with C₁₋₆ alkyl; amido, (loweralkyl)amide; or B is an acyl derivative of formula R₄—C(O)— wherein R₄is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, hydroxy or C₁₋₆alkoxy; or

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ a alkylcycloalkyl, both optionallysubstituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl; or

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy; or

(v) Het optionally substituted with C₁₋₆ alkyl, hydroxy, amido or amino;

or B is a carboxyl of formula R₄—O—C(O)—, wherein R₄ is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy or amido, (lower alkyl)amide, amino optionallymono- or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl, C₄₋₁₀ alkylcycloalkyl, all optionally substitutedwith carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (lower alkyl)amide, aminooptionally mono- or di-substituted with C₁₋₆ alkyl; or

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amino optionally substituted with C₁₋₆ alkyl; or

(v) Het or (lower alkyl)-Het, both optionally substituted with C₁₋₆alkyl, hydroxy, amido, or amino optionally mono-substituted with C₁₋₆alkyl;

or B is an amide of formula R₄—N(R₅)—C(O)— wherein R₄ is

(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amido, (lower alkyl)amide, amino optionally mono-or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, amino optionally mono- or di-substituted with C₁₋₆ alkyl;and R₅ is H or methyl; or

R₄ is (iii) amino optionally mono- or di-substituted with C₁₋₃ alkyl; or

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amino or amido optionally substituted with C₁₋₆alkyl; or

(v) Het optionally substituted with C₁₋₆ alkyl, hydroxy, amino or amido;or

B is a thioamide of formula R₄—NH—C(S)—; wherein R₄ is:

(i) C₁₋₁₀ alkyl; or (ii) C₃₋₇ cycloalkyl; or

B is an amide of formula R₄—NH—C(O)— wherein R₄ is

i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy amido, (lower alkyl)amide, amino optionally mono-or di-substituted with C₁₋₆ alkyl;

(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, amino optionally mono- or di-substituted with C₁₋₆ alkyl;

(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl optionally substituted with C₁₋₆alkyl, hydroxy, amino or amido;

Y is H;

R³ is the side chain of tert-butylglycine (Tbg), Ile, Val, Chg or:

R² is 1-naphtylmethoxy; or quinolinoxy unsubstituted, mono- ordi-substituted with

R₂₁ as defined above, or

R² is:

wherein R_(21A) is C₁₋₆ alkyl; C₁₋₆ alkoxy; C₆, C₁₀ aryl or Het; lowerthioalkyl; halo; amino optionally mono-substituted with C₁₋₆ alkyl; orC₆,C₁₀ aryl, C₇₋₁₆ aralkyl or Het, optionally substituted with R₂₂wherein R₂₂ is C₁₋₆ alkyl, C₁₋₆ alkoxy, amido, (lower alkyl)amide, aminooptionally mono- or di-substituted with C₁₋₆ alkyl, or Het; P1 is acyclopropyl ring wherein carbon 1 has the R configuration,

and R¹ is ethyl, vinyl, cyclopropyl, 1 or 2-bromoethyl or 1 or2-bromovinyl.

Further included in the scope of the invention are compounds of formulaI wherein:

B is tert-butoxycarbonyl (Boc) or

R³ is the side chain of Tbg, Chg or Val;

R² is:

wherein R_(22A) is C₁₋₆ alkyl (such as methyl); C₁₋₆ alkoxy (such asmethoxy); or halo (such as chloro); R_(22B) is C₁₋₆ alkyl, aminooptionally mono-substituted with C₁₋₆ alkyl, amido, or (loweralkyl)amide; and R_(21B) is C₁₋₆ alkyl, C₁₋₆alkoxy, amino, di(loweralkyl)amino, (lower alkyl)amide, NO₂, OH, halo, trifluoromethyl, orcarboxyl; and P1 is:

Finally, included within the scope of this invention is each compound offormula I as presented in Tables 1 to 10.

According to an alternate embodiment, the pharmaceutical compositions ofthis invention may additionally comprise another anti-HCV agent.Examples of anti-HCV agents include, α- or β-interferon, ribavirin andamantadine.

According to another alternate embodiment, the pharmaceuticalcompositions of this invention may additionally comprise otherinhibitors of HCV protease.

According to yet another alternate embodiment, the pharmaceuticalcompositions of this invention may additionally comprise an inhibitor ofother targets in the HCV life cycle, including but not limited to,helicase, polymerase, metalloprotease or internal ribosome entry site(IRES).

The pharmaceutical compositions of this invention may be administeredorally, parenterally or via an implanted reservoir. Oral administrationor administration by injection is preferred. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, and intralesional injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example Tween 80) and suspending agents.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

Other suitable vehicles or carriers for the above noted formulations andcompositions can be found in standard pharmaceutical texts, e.g. in“Remington's Pharmaceutical Sciences”, The Science and Practice ofPharmacy, 19^(th) Ed. Mack Publishing Company, Easton, Pa., (1995).

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably between about 0.5 and about 75 mg/kg body weight per dayof the protease inhibitor compounds described herein are useful in amonotherapy for the prevention and treatment of HCV mediated disease.Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to about 5 times per day or alternatively, asa continuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Preferably, such preparations containfrom about 20% to about 80% active compound.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of theinfection, the patient's disposition to the infection and the judgmentof the treating physician. Generally, treatment is initiated with smalldosages substantially less than the optimum dose of the peptide.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. In general, thecompound is most desirably administered at a concentration level thatwill generally afford antivirally effective results without causing anyharmful or deleterious side effects.

When the compositions of this invention comprise a combination of acompound of formula I and one or more additional therapeutic orprophylactic agent, both the compound and the additional agent should bepresent at dosage levels of between about 10 to 100%, and morepreferably between about 10 and 80% of the dosage normally administeredin a monotherapy regimen.

When these compounds or their pharmaceutically acceptable salts areformulated together with a pharmaceutically acceptable carrier, theresulting composition may be administered in vivo to mammals, such asman, to inhibit HCV NS3 protease or to treat or prevent HCV virusinfection. Such treatment may also be achieved using the compounds ofthis invention in combination with agents which include, but are notlimited to: immunomodulatory agents, such as α-, β-, or γ-interferons;other antiviral agents such as ribavirin, amantadine; other inhibitorsof HCV NS3 protease; inhibitors of other targets in the HCV life cycle,which include but not limited to, helicase, polymerase, metalloprotease,or internal ribosome entry site (IRES); or combinations thereof. Theadditional agents may be combined with the compounds of this inventionto create a single dosage form. Alternatively these additional agentsmay be separately administered to a mammal as part of a multiple dosageform.

Accordingly, another embodiment of this invention provides methods ofinhibiting HCV NS3 protease activity in mammals by administering acompound of the formula I, wherein the substituents are as definedabove.

In a preferred embodiment, these methods are useful in decreasing HCVNS3 protease activity in a mammal. If the pharmaceutical compositioncomprises only a compound of this invention as the active component,such methods may additionally comprise the step of administering to saidmammal an agent selected from an immunomodulatory agent, an antiviralagent, a HCV protease inhibitor, or an inhibitor of other targets in theHCV life cycle such as helicase, polymerase, or metallo protease orIRES. Such additional agent may be administered to the mammal prior to,concurrently with, or following the administration of the compositionsof this invention.

In an alternate preferred embodiment, these methods are useful forinhibiting viral replication in a mammal. Such methods are useful intreating or preventing HCV disease. If the pharmaceutical compositioncomprises only a compound of this invention as the active component,such methods may additionally comprise the step of administering to saidmammal an agent selected from an immunomodulatory agent, an antiviralagent, a HCV protease inhibitor, or an inhibitor of other targets in theHCV life cycle. Such additional agent may be administered to the mammalprior to, concurrently with, or following the administration of thecomposition according to this invention.

The compounds set forth herein may also be used as laboratory reagents.The compounds of this invention may also be used to treat or preventviral contamination of materials and therefore reduce the risk of viralinfection of laboratory or medical personnel or patients who come incontact with such materials (e.g. blood, tissue, surgical instrumentsand garments, laboratory instruments and garments, and blood collectionapparatuses and materials).

The compounds set forth herein may also be used as research reagents.The compounds of this invention may also be used as positive control tovalidate surrogate cell-based assays or in vitro or in vivo viralreplication assays.

Process

The compounds of the present invention were synthesized according to ageneral process as illustrated in scheme I (wherein CPG is a carboxylprotecting group and APG is an amino protecting group):

Briefly, the P1, P2, and P3 can be linked by well known peptide couplingtechniques. The P1, P2, and P3 groups may be linked together in anyorder as long as the final compound corresponds to peptides of FormulaI. For example, P3 can be linked to P2-P1; or P1 linked to P3-P2.

Generally, peptides are elongated by deprotecting the α-amino group ofthe N-terminal residue and coupling the unprotected carboxyl group ofthe next suitably N-protected amino acid through a peptide linkage usingthe methods described. This deprotection and coupling procedure isrepeated until the desired sequence is obtained. This coupling can beperformed with the constituent amino acids in stepwise fashion, asdepicted in Scheme I, or by solid phase peptide synthesis according tothe method originally described in Merrifield, J. Am. Chem. Soc.,(1963), 85, 2149-2154, the disclosure of which is hereby incorporated byreference. Coupling between two amino acids, an amino acid and apeptide, or two peptide fragments can be carried out using standardcoupling procedures such as the azide method, mixed carbonic-carboxylicacid anhydride (isobutyl chloroformate) method, carbodiimide(dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-solublecarbodiimide) method, active ester (p-nitrophenyl ester,N-hydroxysuccinic imido ester) method, Woodward reagent K-method,carbonyldiimidazole method, phosphorus reagents or oxidation-reductionmethods. Some of these methods (especially the carbodiimide method) canbe enhanced by adding 1-hydroxybenzotriazole. These coupling reactionscan be performed in either solution (liquid phase) or solid phase.

More explicitly, the coupling step involves the dehydrative coupling ofa free carboxyl of one reactant with the free amino group of the otherreactant in the presence of a coupling agent to form a linking amidebond. Descriptions of such coupling agents are found in generaltextbooks on peptide chemistry, for example, M. Bodanszky, “PeptideChemistry”, 2^(nd) rev ed., Springer-Verlag, Berlin, Germany, (1993).Examples of suitable coupling agents are N,N′-dicyclohexylcarbodiimide,1-hydroxybenzotriazole in the presence of N,N′-dicyclohexylcarbodiimideor N-ethyl-N′-[(3-dimethylamino)propyl]carbodiimide. A practical anduseful coupling agent is the commercially available(benzotriazol-1-yloxy)tris-(dimethylamino)phosphoniumhexafluorophosphate, either by itself or in the presence of1-hydroxybenzotriazole. Another practical and useful coupling agent iscommercially available2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.Still another practical and useful coupling agent is commerciallyavailable O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate.

The coupling reaction is conducted in an inert solvent, e.g.dichloromethane, acetonitrile or dimethylformamide. An excess of atertiary amine, e.g. diisopropylethylamine, N-methylmorpholine orN-methylpyrrolidine, is added to maintain the reaction mixture at a pHof about 8. The reaction temperature usually ranges between 0° C. and50° C. and the reaction time usually ranges between 15 min and 24 h.

When a solid phase synthetic approach is employed, the C-terminalcarboxylic acid is attached to an insoluble carrier (usuallypolystyrene). These insoluble carriers contain a group that will reactwith the carboxylic group to form a bond that is stable to theelongation conditions but readily cleaved later. Examples of which are:chloro- or bromomethyl resin, hydroxymethyl resin, trytil resin and2-methoxy-4-alkoxy-benzylaloconol resin.

Many of these resins are commercially available with the desiredC-terminal amino acid already incorporated. Alternatively, the aminoacid can be incorporated on the solid support by known methods (Wang,S.-S., J. Am. Chem. Soc., (1973), 9, 1328; Atherton, E.; Shepard, R. C.“Solid-phase peptide synthesis; a practical approach” IRL Press: Oxford,(1989); 131-148). In addition to the foregoing, other methods of peptidesynthesis are described in Stewart and Young, “Solid Phase PeptideSynthesis”, 2^(nd) ed., Pierce Chemical Co., Rockford, Ill. (1984);Gross, Meienhofer, Udenfriend, Eds., “The Peptides: Analysis, Synthesis,Biology”, Vol. 1, 2, 3, 5, and 9, Academic Press, New-York, (1980-1987);Bodansky et al., “The Practice of Peptide Synthesis” Springer-Verlag,New-York (1984), the disclosures of which are hereby incorporated byreference.

The functional groups of the constituent amino acids generally must beprotected during the coupling reactions to avoid formation of undesiredbonds. The protecting groups that can be used are listed in Greene,“Protective Groups in Organic Chemistry”, John Wiley & Sons, New York(1981) and “The Peptides: Analysis, Synthesis, Biology”, Vol. 3,Academic Press, New York (1981), the disclosures of which are herebyincorporated by reference.

The α-carboxyl group of the Cterminal residue is usually protected as anester (CPG) that can be cleaved to give the carboxylic acid. Protectinggroups that can be used include: 1) alkyl esters such as methyl,trimethylsilylethyl and t-butyl, 2) aralkyl esters such as benzyl andsubstituted benzyl, or 3) esters that can be cleaved by mild basetreatment or mild reductive means such as trichloroethyl and phenacylesters.

The α-amino group of each amino acid to be coupled to the growingpeptide chain must be protected (APG). Any protecting group known in theart can be used. Examples of such groups include: 1) acyl groups such asformyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromaticcarbamate groups such as benzyloxycarbonyl (Cbz or Z) and substitutedbenzyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3)aliphatic carbamate groups such as tert-butyloxycarbonyl (Boc),ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4)cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl andadamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl andbenzyl; 6) trialkylsilyl such as trimethylsilyl; and 7) thiol containinggroups such as phenylthiocarbonyl and dithiasuccinoyl. The preferredα-amino protecting group is either Boc or Fmoc. Many amino acidderivatives suitably protected for peptide synthesis are commerciallyavailable.

The α-amino protecting group of the newly added amino acid residue iscleaved prior to the coupling of the next amino acid. When the Boc groupis used, the methods of choice are trifluoroacetic acid, neat or indichloromethane, or HCl in dioxane or in ethyl acetate. The resultingammonium salt is then neutralized either prior to the coupling or insitu with basic solutions such as aqueous buffers, or tertiary amines indichloromethane or acetonitrile or dimethylformamide. When the Fmocgroup is used, the reagents of choice are piperidine or substitutedpiperidine in dimethylformamide, but any secondary amine can be used.The deprotection is carried out at a temperature between 0° C. and roomtemperature (RT) usually 20-22° C.

Any of the amino acids having side chain functionalities must beprotected during the preparation of the peptide using any of theabove-described groups. Those skilled in the art will appreciate thatthe selection and use of appropriate protecting groups for these sidechain functionalities depend upon the amino acid and presence of otherprotecting groups in the peptide. The selection of such protectinggroups is important in that the group must not be removed during thedeprotection and coupling of the α-amino group.

For example, when Boc is used as the α-amino protecting group, thefollowing side chain protecting group are suitable: p-toluenesulfonyl(tosyl) moieties can be used to protect the amino side chain of aminoacids such as Lys and Arg; acetamidomethyl, benzyl (Bn), ort-butylsulfonyl moieties can be used to protect the sulfide containingside chain of cysteine; benzyl (Bn) ethers can be used to protect thehydroxy containing side chains of serine, threonine or hydroxyproline;and benzyl esters can be used to protect the carboxy containing sidechains of aspartic acid and glutamic acid.

When Fmoc is chosen for the α-amine protection, usually tert-butyl basedprotecting groups are acceptable. For instance, Boc can be used forlysine and arginine, tert-butyl ether for serine, threonine andhydroxyproline, and tert-butyl ester for aspartic acid and glutamicacid. Triphenylmethyl (Trityl) moiety can be used to protect the sulfidecontaining side chain of cysteine.

Once the elongation of the peptide is completed all of the protectinggroups are removed. When a liquid phase synthesis is used, theprotecting groups are removed in whatever manner is dictated by thechoice of protecting groups. These procedures are well known to thoseskilled in the art.

When a solid phase synthesis is used, the peptide is cleaved from theresin simultaneously with the removal of the protecting groups. When theBoc protection method is used in the synthesis, treatment with anhydrousHF containing additives such as dimethyl sulfide, anisole, thioanisole,or p-cresol at 0° C. is the preferred method for cleaving the peptidefrom the resin. The cleavage of the peptide can also be accomplished byother acid reagents such as trifluoromethanesulfonicacid/trifluoroacetic acid mixtures. If the Fmoc protection method isused, the N-terminal Fmoc group is cleaved with reagents describedearlier. The other protecting groups and the peptide are cleaved fromthe resin using solution of trifluoroacetic acid and various additivessuch as anisole, etc.

1. Synthesis of Capping Group B

Different capping groups B are introduced in the following manner:

1.1) When B is an aryl, aralkyl: the arylated amino acids were preparedby one of the three methods below:

a) Direct nucleophilic displacement on a fluoro-nitro aryl moiety:

Briefly, 4-fluoro-3-nitrobenzotrifluoride (a) was reacted with L-aminoacid (b) in the presence of a base such as potassium carbonate at 80° C.to yield the desired N-aryl amino acid (c);

b) Copper catalyzed couplings according to Ma et al. (J. Am. Chem. Soc.1998, 120, 12459-12467):

Briefly, bromo-4-fluorobenzene (d) was reacted with L-amino acid (b) inthe presence of a base such as potassium carbonate and a catalyticamount of copper iodide at 90° C. to yield the desired N-aryl amino acid(e); or

c) Nucleophilic displacement of a triflate by an aniline:

Briefly, o-anisidine (e was reacted with triflate (g) in the presence ofa base such as 2,6-lutidine at 90° C. to give benzyl ester (h).Hydrogenation with 10% Pd/C yielded the desired N-aryl amino acid (i).

1.2) When B is an aminothiazole derivative:

a) The Fmoc-thiocyanate prepared according to Kearney et al., 1998, J.Org. Chem, 63, 196, was reacted with a protected P3 residue or the wholepeptide or a peptide segment to provide the thiourea.

b) The thiourea derivative is reacted with an appropriate bromoketone toprovide the corresponding thiazole derivative.

1.3) When B is R₄—C(O)—, R₄—S(O)₂:

Protected P3 or the whole peptide or a peptide segment is coupled to anappropriate acyl chloride or sulfonyl chloride respectively, that iseither commercially available or for which the synthesis is well knownin the art.

1.4) When B is R₄O—C(O)—:

Protected P3 or the whole peptide or a peptide segment is coupled to anappropriate chloroformate that is either commercially available or forwhich the synthesis is well known in the art. For Boc-derivatives(Boc)₂O is used.

For example:

a) Cyclobutanol is treated with phosgene to furnish the correspondingchloroformate.

b) The chloroformate is treated with the desired NH₂-tripeptide in thepresence of a base such as triethylamine to afford thecyclobutylcarbamate.

1.5) When B is R₄—N(R₅)—C(O)—, or R₄—NH—C(S)—, protected P3 or the wholepeptide or a peptide segment is treated with phosgene followed by amineas described in SynLett. Feb 1995; (2); 142-144

2. Synthesis of P2 Moieties

2.1 Synthesis of precursors:

A) Synthesis of haloarylmethane derivatives.

The preparation of halomethyl-8-quinoline IId was done according to theprocedure of K. N. Campbell et al., J. Amer. Chem. Soc., (1946), 68,1844.

Briefly, 8-quinoline carboxylic acid IIa was converted to thecorresponding alcohol IIc by reduction of the corresponding acyl halideIIb with a reducing agent such as lithium aluminium hydride. Treatmentof alcohol IIb with the appropriate hydrohaloacid gives the desired haloderivative IId. A specific embodiments of this process is presented inExample 1.

B) Synthesis of aryl alcohol derivatives:

2-phenyl-4-hydroxyquinoline derivatives IIc were prepared according toGiardina et al. (J. Med. Chem., (1997), 40, 1794-1807).

R₂₂ & R_(21B)=alkyl, OH, SH, halo, NH₂, NO₂.

Briefly, benzoylacetamide (IIIa) was condensed with the appropriateaniline (IIIb) and the imine obtained was cyclized with polyphosphoricacid to give the corresponding 2-phenyl-4-hydroxyquinoline (IIIc). Aspecific embodiment of this process is presented in Example 2.

Or alternatively, the process can be carried out in a different manner:Benzoylethyl ester (IIIa) was condensed with the appropriate aniline(IIIb) in the presence of acid and the imine obtained was cyclized byheating at 260-280° C. to give the corresponding2-phenyl-4-hydroxyquinoline (IIIc). A specific embodiments of thisprocess is presented in Example 3 (compound 3e).

2.2. Synthesis of P2:

A) The synthesis of 4-substituted proline (wherein R² is attached to thering via a carbon atom) (with the stereochemistry as shown):

 is done as shown in Scheme IV according to the procedures described byJ. Ezquerra et al. (Tetrahedron, (1993), 38, 8665-8678) and C. Pedregalet al. (Tetrahedron Lett., (1994), 35, 2053-2056).

Briefly, Boc-pyroglutamic acid is protected as a benzyl ester. Treatmentwith a strong base such as lithium diisopropylamide followed by additionof an alkylating agent (Br—R²⁰ or I—R²⁰) gives the desired compounds IVeafter reduction of the amide and deprotection of the ester.

B) The synthesis of O-substituted-4-(R)-hydroxyproline:

 may be carried out using the different processes described below.

1) When R²⁰ is aryl, aralkyl, Het or (lower alkyl)-Het, the process canbe carried out according to the procedure described by E. M. Smith etal. (J. Med. Chem. (1988), 31, 875-885). Briefly, commercially availableBoc-4(R)-hydroxyproline is treated with a base such as sodium hydride orpotassium tert-butoxide and the resulting alkoxide reacted with halo-R²⁰(Br—R²⁰, I—R²⁰, etc.) to give the desired compounds. Specificembodiments of this process are presented in Examples 4, 5 and 7.

2) Alternatively, when R²⁰ is aryl or Het, the compounds can also beprepared via a Mitsunobu reaction (Mitsunobu (1981), Synthesis, January,1-28; Rano et al., (1995), Tet. Lett. 36(22), 3779-3792; Krchnak et al.,(1995), Tet. Lett. 36(5), 62193-6196; Richter et al., (1994), Tet. Left.35(27), 4705-4706). Briefly, commercially availableBoc-4(S)-hydroxyproline methyl ester is treated with the appropriatearyl alcohol or thiol in the presence of triphenylphosphine anddiethylazodicarboxylate (DEAD) and the resulting ester is hydrolyzed tothe acid. Specific embodiments of this process are presented in Examples6 and 8.

Alternatively, the Mitsunobu reaction can be carried out in solid phase(Scheme V). The 96-well block of the Model 396 synthesizer (advancedChemTech) is provided with aliquots of resin-bound compound (Va) and avariety of aryl alcohols or thiols and appropriate reagents are added.After incubation, each resin-bound product (Vb) is washed, dried, andcleaved from the resin.

A Suzuki reaction (Miyaura et al., (1981), Synth. Comm. 11, 513; Sato etal., (1989), Chem. Left., 1405; Watanabe et al., (1992), Synlett., 207;Takayuki et al., (1993), J. Org. Chem. 58, 2201; Frenette et al.,(1994), Tet. Left. 35(49), 9177-9180; Guiles et al., (1996), J. Org.Chem. 61, 5169-5171) can also be used to further functionalize the arylsubstituent.

3. Synthesis of P1 moieties

3.1 Synthesis of the 4 possible isomers of 2-substituted1-aminocyclopropyl carboxylic acid

The synthesis was done according to scheme VI.

a) Briefly, di-protected malonate VIa and 1,2-dihaloalkane VIb or cyclicsulfate VIc (synthesized according to K. Burgess and Chun-Yen KE(Synthesis, (1996), 1463-1467) are reacted under basic conditions togive the diester VId.

b) A regioselective hydrolysis of the less hindered ester is performedto give the acid VIe.

c) This acid VIe is subjected to a Curtius rearrangement to give aracemic mixture of 1-aminocyclopropylcarboxylic acid derivatives VIfwith R¹ being syn to the carboxyl group. A specific embodiment for thissynthesis is presented in Example 9.

d, e) Alternatively, selective ester formation from the acid VIe with anappropriate halide (P*Cl) or alcohol (P*OH) forms diester VIg in whichthe P* ester is compatible with the selective hydrolysis of the P ester.Hydrolysis of P ester provides acid VIh.

f) A Curtius rearrangement on VIh gives a racemic mixture of1-aminocyclopropylcarboxylic acid derivatives VIi with R¹ group beinganti to the carboxyl group. A specific embodiment for this synthesis ispresented in Example 14.

An alternative synthesis for the preparation of derivatives VIIf (whenR¹ is vinyl and syn to the carboxyl group) is described below.

Treatment of commercially available or easily obtainable imines VIIawith 1,4-dihalobutene VIIb in presence of a base produces, afterhydrolysis of the resulting imine VIIc, VIId having the allylsubstituent syn to the carboxyl group. Specific embodiments of thisprocess are presented in Example 15 and 19.

Resolution of all of the above enantiomeric mixtures at carbon 1 (VIeand VIId) can be carried out via:

1) enzymatic separation (Examples 13, 17 and 20);

2) crystallization with a chiral acid (Example 18); or

3) chemical derivatization (Example 10).

Following resolution, determination of the absolute stereochemistry canbe carried out as presented in Example 11.

Enantiomeric resolution and stereochemistry determination can be carriedout in the same manner for the enantiomeric mixtures at carbon 1 whereinthe substituent at C2 is anti to the carboxyl group (VIi).

3.2 Synthesis of 1-aminocyclobutyl carboxylic acid

The synthesis of 1,1-aminocyclobutanecarboxylic acid is carried outaccording to “Kavin Douglas; Ramaligam Kondareddiar; Woodard Ronald,Synth. Commun. (1985), 15 (4), 267-72.

Briefly, treatment of compound VIIIa with a base in the presence ofVIIIb gives the corresponding cyclobutyl derivative VIIIc. Hydrolysis ofthe isocyanate and ester groups of VIIIc under acidic conditions (HCl)yields the hydrochloride salt of the 1-amino-cyclobutylcarboxylic acidVIIId. The carboxylic acid is later esterified under methanol in HCl. Aspecific embodiment of this esterification is described in Example 21.

3.3 Synthesis of 2-substituted 1-aminocyclobutyl carboxylic acid

a) A protected glycine ester derivative such as imine IXa is alkylatedwith an homoallylic electrophile IXb using an appropriate base such as ametal hydride, hydroxide or alkoxide. Useful leaving groups in IXbinclude halogens (X=Cl, Br, I) or sulfonate esters (mesylate, tosylateor triflate). The allylic alcohol functionality in IXb is protected withhydroxyl protecting groups well known in the art (e.g. acetate, silyl,acetals).

b) In a second step, the hydroxyl function of monoalkylated derivativeIXc is de-protected and converted to a suitable electrophilic function Xsuch as described above for compound IXb.

c) Cyclization of IXd to cyclobutane derivative IXe is carried out bytreatment with a base (metal hydrides, alkoxides), followed byhydrolysis using aqueous mineral acids and neutralization with a mildbase. At this stage, syn and anti-isomers of IXe can be separated byflash chromatography.

d) Optionally, the double bond in IXe can also be hydrogenated understandard conditions to yield the corresponding saturated derivative IXf.

The invention further comprises a process for the preparation of apeptide analog of formula (I) wherein P1 is a substitutedaminocyclopropyl carboxylic acid residue, comprising the step of:

coupling a peptide selected from the group consisting of: APG-P3-P2; orAPG-P2;

with a P1 intermediate of formula:

wherein R¹ is C₁₋₆ alkyl, cycloalkyl or C₂₋₆ alkenyl, all optionallysubstituted with halogen, CPG is a carboxyl protecting group and APG isan amino protecting group and P3 and P2 are as defined above.

The invention further comprises a process for the preparation of: 1)aserine protease inhibitor peptide analog, or 2) a HCV NS3 proteaseinhibitor peptide analog, this process comprising the step of:

coupling a (suitably protected) amino acid, peptide or peptide fragmentwith a P1 intermediate of formula:

wherein R¹ is C₁₋₆ alkyl, C₃₋₇ cycloalkyl or C₂₋₆ alkenyl, alloptionally substituted with halogen, and CPG is a carboxyl protectinggroup.

The invention therefore comprises a process for the preparation of: 1) aprotease inhibitor peptide analog, or 2) a serine protease inhibitorpeptide analog, this process comprising the step of:

coupling a (suitably protected) amino acid, peptide or peptide fragmentwith an intermediate of formula:

wherein CPG is a carboxyl protecting group.

The invention also comprises the use of a P1 intermediate of formula:

wherein R¹ is C₁₋₆ alkyl, cycloalkyl or C₂₋₆ alkenyl, all optionallysubstituted with halogen, for the preparation of: 1)a serine proteaseinhibitor peptide analog, or 2) a HCV NS3 protease inhibitor peptideanalog.

The invention also comprises the use of an intermediate of formula:

wherein CPG is a carboxyl protecting group, for the preparation of: 1) aprotease inhibitor peptide analog, or 2) a serine protease inhibitorpeptide analog.

The invention also comprises the use of a P1 intermediate of formula:

wherein R¹ is C₁₋₆ alkyl, cycloalkyl or C₂₋₆ alkenyl, all optionallysubstituted with halogen, for the preparation of a compound of formula Ias defined above.

Finally, the invention also comprises the use of a proline analog offormula:

wherein

R_(21A) is C₁₋₆ alkyl; C₁₋₆ alkoxy; lower thioalkyl; halo; aminooptionally mono-substituted with C₁₋₆ alkyl; C₆, C₁₀ aryl, C₇₋₁₆ aralkylor Het, said aryl, aralkyl or Het optionally substituted with R₂₂wherein R₂₂ is C₁₋₆ alkyl, C₁₋₆ alkoxy, amido, (lower alkyl)amide, aminooptionally mono- or di-substituted with C₁₋₆ alkyl, or Het, and

R_(21B) is C₁₋₆ alkyl, C₁₋₆ alkoxy, amino, di(lower alkyl)amino, (loweralkyl)amide, NO₂, OH, halo, trifluoromethyl, or carboxyl;

for the synthesis of 1) a serine protease inhibitor peptide analog, 2) aHCV NS3 protease inhibitor peptide analog, or 3) a peptide analog offormula I as defined above.

EXAMPLES

The present invention is illustrated in further detail by the followingnon-limiting examples.

Temperatures are given in degrees Celsius. Solution percentages expressa weight to volume relationship, and solution ratios express a volume tovolume relationship, unless stated otherwise. Nuclear magnetic resonance(NMR) spectra were recorded on a Bruker 400 MHz spectrometer; thechemical shifts (δ) are reported in parts per million. Flashchromatography was carried out on silica gel (SiO₂) according to Still'sflash chromatography technique (W. C. Still et al., J. Org. Chem.,(1978), 43, 2923).

Abbreviations used in the examples include Bn: benzyl; Boc:tert-butyloxycarbonyl {Me₃COC(O)}; BSA: bovine serum albumin; CHAPS:3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; DBU:1,8-diazabicyclo[5.4.0]undec-7-ene; CH₂Cl₂=DCM: methylene chloride;DEAD: diethylazodicarboxylate; DIAD: diisopropylazodicarboxylate; DIEA:diisopropylethylamine; DIPEA: diisopropylethylamine; DMAP:dimethylaminopyridine; DCC: 1,3-dicyclohexylcarbodiimide; DME:1,2-dimethyoxyethane; DMF: dimethylformamide; DMSO: dimethylsulfoxide;DTT: dithiothreitol or threo-1,4-dimercapto-2,3-butanediol; DPPA:diphenylphosphoryl azide; EDTA: ethylenediaminetetraacetic acid; Et:ethyl; EtOH: ethanol; EtOAc: ethyl acetate; Et₂O: diethyl ether; HATU:[O-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate]; HPLC: high performance liquid chromatography; MS:mass spectrometry (MALDI-TOF: Matrix Assisted Laser DisorptionIonization-Time of Flight, FAB: Fast Atom Bombardment); LAH: lithiumaluminum hydride; Me: methyl; MeOH: methanol; MES:(2-{N-morpholino}ethane-sulfonic acid); NaHMDS: sodiumbis(trimethylsilyl)amide; NMM: N-methylmorpholine; NMP:N-methylpyrrolidine; Pr: propyl; Succ: 3-carboxypropanoyl; PNA:4-nitrophenylamino or p-nitroanilide; TBAF: tetra-n-butylammoniumfluoride; TBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate; TCEP: tris(2-carboxyethyl) phosphine hydrochloride;TFA: trifluoroacetic acid; THF: tetrahydrofuran; TIS:triisopropylsilane; TLC: thin layer chromatography; TMSE:trimethylsilylethyl; Tris/HCl: tris(hydroxymethyl)aminomethanehydrochloride.

P2 Building Blocks Example 1 Synthesis of bromomethyl-8quinoline (1)

To commercially available 8-quinoline carboxylic acid (2.5 g, 14.4 mmol)was added neat thionyl chloride (10 ml, 144 mmol). This mixture washeated at 80° C. for 1 h before the excess thionyl chloride wasdistilled off under reduced pressure. To the resulting brownish solidwas added absolute EtOH (15 mL) which was heated at 80° C. for 1 hbefore being concentrated in vacuo. The residue was partitioned betweenEtOAc and saturated aqueous NaHCO₃, and the organic phase dried (MgSO₄),filtered and concentrated to give a brownish oil (2.8 g). This material(ca. 14.4 mmol) was added dropwise over 35 min to a LAH (0.76 g, 20.2mmol)/Et₂O suspension which was cooled to −60° C. The reaction mixturewas slowly warmed to −35° C. over 1.5 h before the reaction wascomplete. The reaction was quenched with MgSO₄.10H₂O slowly over 30 minand then wet THF. The mixture was partitioned between Et₂O and 10%aqueous NaHCO₃.The organic phase was dried (MgSO₄), filtered andconcentrated to give a yellowish solid (2.31 g, 80% over 2 steps)corresponding to the alcohol. The alcohol (2.3 g, 11.44 mmol) wasdissolved in AcOH/HBr (20 mL, 30% solution from Aldrich) and heated at70° C. for 2.5 h. The mixture was concentrated in vacuo to dryness,partitioned between EtOAc (100 mL) and saturated aqueous NaHCO₃ beforebeing dried (MgSO₄), filtered and concentrated to give the desiredcompound (1) as a brownish solid (2.54 g, 100%).

Example 2 Synthesis of 2-phenyl-4-hydroxyquinoline (2)

Commercially available ethyl benzoylacetate (6.00 g, 31.2 mmol) washeated at 85° C. (sealed tube) in 75 mL of 30% NH₄OH for 2 hours. Thesolid formed upon cooling was filtered and refluxed in water for 2hours. The solution was extracted three times with CH₂Cl₂. The organiclayers were combined, dried over MgSO₄, filtered and concentrated. Theyellow residue was flash chromatographed on silica gel, eluting withEtOAc:hexane (3:7), to give the corresponding amide as a white solid,1.60 g, 31% yield.

This amide (250 mg, 1.53 mmol) was refluxed using a Dean-Stark apparatuswith aniline (143 mg, 1.53 mmol) and aniline HCl (10 mg, 0.08 mmol) intoluene (10 mL) for 16 h. The solution was concentrated to afford abrown oil that was mixed with polyphosphoric acid (2 g) and heated at135° C. for 20 min. The reaction mixture was poured into water andadjusted to pH 8 with 5 M NaOH. The aqueous suspension was extractedtwice with ethyl acetate. The organic layers were combined, washed withbrine, dried over MgSO₄, filtered and concentrated. The residue wasflash chromatographed on silica gel, eluting with 3% MeOH in ethylacetate, to give 2-phenyl-4-hydroxyquinoline (2), 67 mg, 20% yield.

¹H NMR (DMSO-d₆) δ8.11 (d, J=7 Hz, 1 H), 7.86-7.83 (m, 2 H), 7.77 (d,J=8 Hz, 1 H), 7.68 (dd, J=8,7 Hz, 1 H), 7.61-7.58 (m, 3 H), 7.35 (dd,J=8, 7 Hz, 1 H), 6.34 (s, 1 H).

Example 3 Synthesis of 4-hydroxy-2-phenyl-7-methoxyquinoline (3)

4-hydroxy-2-phenyl-7-methoxyquinoline (e)

A solution of ethyl benzoylacetate (b) (100.0 g, 0.52 mol), m-anisidine(a) (128.1 g, 1.04 mol) and 4 N HCl/dioxane (5.2 mL) in toluene (1.0 L)was refluxed for 6.25 h in a Dean-Stark apparatus. The cooled toluenesolution was successively washed with aqueous 10% HCl (2×300 mL), 1 NNaOH (2×300 mL), H₂O (300 mL) and brine (150 mL). The toluene phase wasdried (MgSO₄), filtered and concentrated under reduced pressure to givea 1.2:1.0 mixture of ester c and amide d (144.6 g, 45%/38% crude yield)as a dark brown oil. The crude oil was heated to 280° C. for 80 minwhile distilling generated EtOH. The cooled dark solid obtained wastriturated with CH₂Cl₂ (200 mL). The suspension was filtered and theresulting solid washed with CH₂Cl₂ to give e (22.6 g, 17% from a) as abeige solid: ¹H NMR (DMSO-d₆) δ8.00 (d, J=9.0 Hz, 1H), 7.81-7.82 (m,2H), 7.57-7.59 (m, 3H), 7.20 (d, J=2.2 Hz, 1H), 6.94 (dd, J=9.0, 2.2 Hz,1H), 6.26 (s, 1H), 3.87 (s, 3H).

4-Chloro-2-phenyl-7-methoxyquinoline (3)

A suspension of e (8.31 g, 33.1 mmol) in POCl₃ (90 mL) was heated toreflux for 2 h (clear solution obtained upon heating). The reactionmixture was concentrated under reduced pressure. The residue waspartitioned between 1 N NaOH (exothermic, 10 N NaOH added to maintainhigh pH) and EtOAc (500 mL). The organic layer was washed with H₂O (100mL) and brine (100 mL) then was dried (MgSO₄), filtered and concentratedunder reduced pressure to give 3 (8.60 g, 96%) as a pale yellow solid:¹H NMR (DMSO-d₆) δ8.28-8.30 (m, 2H), 8.20 (s, 1H), 8.10 (d, J=9.1 Hz,1H), 7.54-7.58 (m, 3H), 7.52 (d, J=2.5 Hz, 1H), 7.38 (dd, J=9.1, 2.5 Hz,1H), 3.98 (s, 3H). This reaction was repeated three times and gavealways 96-98% yield which is significantly higher that the 68% yieldreported in J. Med. Chem. 1997, 40, 1794.

Example 4 Synthesis of Boc-4(R)-(naphthalen-1-ylmethoxy) proline (4)

Commercially available Boc-4(R)-hydroxyproline (5.00 g, 21.6 mmol) wasdissolved in THF (100 mL) and cooled to 0° C. Sodium hydride (60%dispersion in oil, 1.85 g, 45.4 mmol) was added portionwise over 10minutes and the suspension was stirred at RT for 1 h. Then,1-(bromomethyl)naphthalene (8.00 g, 36.2 mmol) (prepared as described inE. A. Dixon et al. Can. J. Chem., (1981), 59, 2629-2641) was added andthe mixture was heated at reflux for 18 h. The mixture was poured intowater (300 mL) and washed with hexane. The aqueous layer was acidifiedwith 10% aqueous HCl and extracted twice with ethyl acetate. The organiclayers were combined and washed with brine, dried (MgSO₄), filtered andconcentrated. The residue was purified by flash chromatography (49:49:2hexane:ethyl acetate:acetic acid) to give the title compound as acolorless oil (4.51 g, 56% yield). ¹H NMR (DMSO-d₆) indicated thepresence of two rotamers: δ8.05 (m, 1H), 7.94 (m, 1H), 7.29 (d, J=14 Hz,1H), 7.55-7.45 (m, 4H), 4.96 (m, 2H), 4.26 (br. s, 1H), 4.12 (dd, J=J=8Hz, 1H), 3.54-3.42 (m, 2H), 2.45-2.34 (m, 1H), 2.07-1.98 (m, 1H) 1.36(s, (3/9) 9H), 1.34 (s, (6/9) 9H).

Example 5 Synthesis of Boc-4(R)-(8-quinoline-methoxy) proline (5)

Boc-4(R)-hydroxyproline (1.96 g, 8.5 mmol) in anhydrous THF (20 mL) wasadded to a suspension of NaH (1.4 g, 60% in oil, 34 mmol) in THF (100mL). This mixture was stirred 30 min before bromomethyl-8-quinoline fromExample 1 (2.54 g, 11.44 mmol) was added in THF (30 mL). The reactionmixture was heated at 70° C. (5 h) before the excess NaH was destroyedcarefully with wet THF. The reaction was concentrated in vacuo and theresulting material was dissolved in EtOAc and H₂O. The basic aqueousphase was separated and acidified with 10% aqueous HCl to pH ˜5 beforebeing extracted with EtOAc (150 mL). The organic phase was dried(MgSO₄), filtered and concentrated to give a brown oil. Purification byflash chromatography (eluent: 10% MeOH/CHCl₃) gave the desired compound(5) as a pale yellow solid (2.73 g, 86%). HPLC (97.5%); ¹H-NMR (DMSO-d₆)shows rotamer populations in a 6:4 ratio, δ12-11.4 (bs, 1H), 8.92 (2×d,J=4.14 and 4.14 Hz, 1H), 8.38 (2×d, J=8.27 and 8.27 Hz, 1H), 7.91 (d,J=7.94 Hz, 1H), 7.77 (d, J=7.0 Hz, 1H), 7.63-7.54 (m, 2H), 5.14 (2×s,2H), 4.32-4.29 (m, 1H), 4.14-4.07 (m, 1H), 3.52-3.44 (m, 2H), 2.43-2.27(m, 1H), 2.13-2.04 (m, 1H), 1.36 and 1.34 (2×s, 9H).

Example 6 Preparation of Boc-4(R)-(7-chloroquinoline-4-oxo)proline (6)

Commercially available Boc-4(S)-hydroxyproline methyl ester (500 mg,2.04 mmol) and 7-chloro-4-hydroxyquinoline (440 mg, 2.45 mmol) wereplaced in dry THF (10 mL) at 0° C. Triphenylphosphine (641 mg, 2.95mmol) was added, followed by slow addition of DIAD (426 mg, 2.45 mmol).The mixture was stirred at RT for 20 h. The reaction mixture was thenconcentrated, taken up in ethyl acetate and extracted three times withHCl 1N. The aqueous phase was basified with Na₂CO₃ and extracted twicewith ethyl acetate. The organic layers were combined, dried over MgSO₄,filtered and concentrated to give a yellow oil. The oil was purified byflash chromatography to give the methyl ester as a white solid, 498 mg,58% yield. This methyl ester (400 mg, 0.986 mmol) was hydrolyzed with 1Maqueous sodium hydroxide (1.7 mL, 1.7 mmol) in methanol (4 mL), at 0°C., for 3 h. The solution was concentrated to remove the methanol andneutralized with 1M aqueous HCl. The suspension was concentrated todryness and taken up in methanol (20 mL), the salts were filtered offand the filtrate concentrated to give the desired compound (6) as awhite solid, 387 mg, quant. yield.

¹H NMR (DMSO-d₆) (ca. 1:1 mixture of rotamers) δ8.74 (d, J=5 Hz, 1 H),8.13-8.09 (m, 1 H), 7.99 and 7.98 (s, 1 H), 7.58 (d, J=9 Hz, 1 H), 7.02(d, J=5 Hz, 1 H), 5.26-5.20 (m, 1 H), 4.10-4.01 (m, 1 H), 3.81-3.72 (m,1 H), 3.59 (dd, J=12, 10 Hz, 1 H), 2.41-2.31 (m, 2 H), 1.34 and 1.31 (s,9H).

Example 7 Synthesis of Boc-4(R)-(2-phenyl-7-methoxyquinoline-4-oxo)proline (7)

Boc-4(R)-(2-phenyl-7-methoxyquinoline-4-oxo) proline (7)

Potassium tert-butoxide (8.16 g, 72.7 mmol) was added in small portions,over 15 min, to a solution of Boc-4(R)-hydroxy proline (6.73 g, 29.1mmol) in DMSO (83 mL) maintained at 25° C. The mixture was stirred at25° C. for 1.5 h. Chloro-2-phenyl-7-methoxyquinoline 3 (8.61 g, 32.0mmol) was added in 4 portions over 15 min to the reaction mixture. Thereaction mixture was stirred at 25° C. for 19 h. The resultingsuspension was poured in H₂O (650 mL) and the mixture was washed withEt₂O (3×150 mL) to remove excess chloroquinoline (EtOAc was later foundto be more efficient). The aqueous layer was acidified with aqueous 1 NHCl (38 mL of calculated 1.5 equiv. required, 43.6 mL) to pH 4-5. Thewhite solid that precipitated was recovered by filtration. The moistsolid was dried under reduced pressure over P₂O₅ to give the prolinederivative 7 (12.6 g, 91%, contains 2.3% w/w of DMSO) as a beige solid:

¹H NMR (DMSO-d 6) δ(2:1 mixture of rotamers) 8.27 (d, J=7.0 Hz, 2H),8.00, 7.98 (2d, J=9.2, ˜9.2 Hz, 1H), 7.48-7.56 (m, 3H), 7.45, 7.43 (2s,1H), 7.39 (d,J=2.5 Hz, 1H), 7.17 (dd, J=9.2, 2.5 Hz, 1H), 5.53-5.59 (m,1H), 4.34-4.41 (m, 1H), 3.93 (s, 3H), 3.76 (broad s, 2H), 2.63-2.73 (m,1H), 2.32-2.43 (m, 1 H), 1.36, 1.33 (2s, 9H).

Example 8 Synthesis of Boc-4(R)-(2-phenyl-6-nitroquinoline-4oxo) proline(8)

Diethyl azodicarboxylate (0.77 mL, 4.89 mmol) was added dropwise to astirred solution of triphenylphosphine (1.28 g, 4.88 mmol) in 15 mL oftetrahydrofuran at 0° C. After 30 min. of stirring under nitrogen asolution of Boc-4(S)-hydroxyproline methyl ester (1.00 g, 4.08 mmol) wasadded in 5 mL of tetrahydrofuran followed by a suspension ofcommercially available 6-nitro-2-phenyl-4-quinolinol (1.30 g, 4.88 mmol)in 10 mL of the same solvent. The red mixture was stirred for 15 min. at0° C. and at RT overnight. The solvent was evaporated in vacuo. Theremaining oil was diluted in ethyl acetate and washed twice with sodiumbicarbonate, once with water and once with brine. The organic layer wasdried (MgSO₄), filtered and evaporated in vacuo. The residue waschromatographed over silica gel (70:30 v/v, hexanes-ethyl acetate)affording the desired methyl ester as a light yellow solid (1.70 9,85%). ¹H NMR(CDCl₃) rotamers ≡3:7 δ9.03 (d, J=2.5 Hz, 1H), 8.46 (dd,J=9, 2.5 Hz, 1H), 8.18 (d, J=9 Hz, 1H), 8.14-8.07 (m, 2H), 7.59-7.50 (m,3H), 7.19 (s, 1H), 5.39-5.30 (m, 1H), 4.67 (t, J=8 Hz, 0.3H), 4.61 (t,J=8 Hz, 0.7H), 4.07-4.01 (m, 2H), 3.81 (s, 3H), 2.89-2.73 (m, 1H),2.55-2.47 (m, 1H), 1.49 (s, 2.7H), 1.45 (s, 6.3H).

To a solution of the methyl ester (503 mg, 1.02 mmol) in a mixture ofTHF: H₂O (10:4 mL) was added lithium hydroxide monohydrate (85 mg, 2.05mmol). 2 mL of MeOH was added in order to get an homogeneous solution. Awhite precipitate resulted within 30 min. The resulting suspension wasstirred at RT for an additional 6 h. The reaction mixture was dilutedwith an aqueous solution of citric acid 10% and extracted with ethylacetate. The organic layer was dried (MgSO₄), filtered and evaporated invacuo to afford 416 mg (85%) of the desired acid (8).

¹H NMR (DMSO-d₆): δ8.92-8.87 (m, 1 H), 8.47 (dd, J=9, 3 Hz, 1H),8.38-8.32 (m, 2H), 8.19 (d, J=9 Hz, 1H), 7.77 (s, 1H), 7.62-7.55 (m,3H), 5.73-5.66 (m,1H), 4.41 (t, J=8 Hz, 1H), 3.89-3.76 (m, 2H),2.83-2.72 (m, 1H), 2.47-2.35 (m, 1H), 1.38 (s, 9H).

P1 Building Blocks Example 9

A) Synthesis of mixture of (1R, 2R)/(1S, 2R) 1-amino-2-ethylcyclopropylcarboxylic acid

a) To a suspension of benzyltriethylammonium chloride (21.0 g, 92.19mmol) in a 50% aqueous NaOH solution (92.4 g in 185 mL H₂O ) weresuccessively added di-tert-butylmalonate (20.0 g, 92.47 mmol) and1,2-dibromobutane (30.0 g, 138.93 mmol). The reaction mixture wasvigorously stirred overnight at RT, a mixture of ice and water was thenadded. The crude product was extracted with CH₂Cl₂ (3×) and sequentiallywashed with water (3×) and brine. The organic layer was dried (MgSO₄),filtered and concentrated. The residue was flash chromatographed (7 cm,2 to 4% Et₂O in hexane) to afford the desired cyclopropane derivative 9c(19.1 g, 70.7 mmol, 76% yield). ¹H NMR (CDCl₃) δ1.78-1.70 (m, 1H), 1.47(s, 9H), 1.46 (s, 9H), 1.44-1.39 (m, 1H), 1.26-1.64 (m, 3H), 1.02 (t,3H, J=7.6 Hz).

b) To a suspension of potassium tert-butoxide (6.71 g, 59.79 mmol, 4.4eq.) in dry ether (100 mL) at 0° C. was added H₂O (270 μL, 15.00 mmol,1.1 eq.). After 5 min diester 9c (3.675 g, 13.59 mmol) in ether (10 mL)was added to the suspension. The reaction mixture was stirred overnightat RT, then poured in a mixture of ice and water and washed with ether(3×). The aqueous layer was acidified with a 10% aq. citric acidsolution at 0° C. and extracted with AcOEt (3×). The combined organiclayer was successively washed with water (2×) and brine. After the usualtreatment (Na₂SO₄, filtration, concentration), the desired acid 9d wasisolated as a pale yellow oil (1.86 g, 8.68 mmol, 64% yield). ¹H NMR(CDCl₃) δ2.09-2.01 (m, 1H), 1.98 (dd, J=3.8, 9.2 Hz, 1H), 1.81-1.70 (m,1H), 1.66 (dd, J=3.0, J=8.2 Hz, 1H), 1.63-1.56 (m, 1H), 1.51 (s, 9H),1.0 (t, J=7.3 Hz, 3H).

c) To the acid 9d (2.017 g, 9.414 mmol) in dry benzene (32 mL) weresuccessively added Et₃N (1.50 mL, 10.76 mmol, 1.14 eq.) and DPPA (2.20mL, 10.21 mmol, 1.08 eq.). The reaction mixture was refluxed for 3.5 hthen 2-trimethylsilylethanol (2.70 mL, 18.84 mmol, 2.0 eq.) was added.The reflux was maintained overnight then the reaction mixture wasdiluted with Et₂O and successively washed with a 10 % aqueous citricacid solution, water, saturated aqueous NaHCO₃, water (2×) and brine.After the usual treatment (MgSO₄, filtration, concentration) the residuewas purified by flash chromatography (5 cm, 10% AcOEt—hexane) to affordthe desired carbamate 9e (2.60 g, 7.88 mmol, 84% yield) as a pale yellowoil. MS (FAB) 330 (MH⁺); ¹H NMR (CDCl₃) δ5.1 (bs, 1H), 4.18-4.13 (m,2H), 1.68-1.38 (m, 4H), 1.45 (s, 9H), 1.24-1.18 (m, 1H), 1.00-0.96 (m,5H), 0.03 (s, 9H).

d) To carbamate 9e (258 mg, 0.783 mmol) was added a 1.0 M TBAF solutionin THF (940 μL, 0.94 mmol, 1.2 eq.). After 4.5 h an additional amount of1.0 M TBAF was added (626 μL, 0.63 mmol, 0.8 eq.). The reaction mixturewas stirred overnight at RT, refluxed for 30 min and then diluted withAcOEt. The solution was successively washed with water (2×) and brine.After the usual treatment (MgSO₄, filtration and concentration) thedesired amine 9f was isolated (84 mg, 0.453 mmol, 58% yield) as a paleyellow liquid. ¹H NMR (CDCl₃) δ1.96 (bs, 2H), 1.60-1.40 (m, 2H), 1.47(s, 9H), 1.31-1.20 (m, 1H), 1.14 (dd, J=4.1, 7.3 Hz, 1H), 1.02 (dd,J=4.1, 9.2 Hz, 1H), 0.94 (t, J=7.3 Hz, 3H).

Example 10 Chemical resolution of t-butyl-(1R, 2R)/(1S, 2R)1-amino-2-ethylcyclopropyl carboxylate (from Example 9)

Compound 9e from Example 9 (8.50 g, 25.86 mmol) was treated with 1MTBAF/THF (26 mL) at reflux for 45 min. The cooled reaction mixture wasdiluted with EtOAc, washed with water (3×) and brine (1×), then, dried(MgSO₄), filtered and evaporated to provide the free amine as a lightyellow oil. The free amine was dissolved in anhydrous CH₂Cl₂ (120 mL),NMM (8.5 mL, 77.57 mmol), compound 4 (Example 4) (10.08 g, 27.15 mmol)and HATU (11.79 g 31.03 mmol) were added successively. The reactionmixture was stirred at RT overnight, then worked up as describedpreviously. The crude diastereomeric mixture was separated by flashchromatography (eluent-hexane: Et₂O; 25:75) to provide the dipeptide 10a(the less polar eluting spot) as a white foam (4.42 g ; 64% of thetheoretical yield) and 10b (the more polar eluting spot) as an ivoryfoam (4 g., 57% of theoretical yield). At this time both isomers wereseparated but the absolute stereochemistry was still not known.

Example 11 Determination of the absolute stereochemistry of compounds10a and 10b by correlation with known t-butyl(1R-amino-2R-ethylcyclopropyl carboxylate

Prof. A. Charette from the University of Montreal, provided compound 11ahaving the absolute stereochemistry as shown, which was determined byX-ray crystallography (J. Am. Chem. Soc., 1995, 117, 12721). Compound11a (13.2 mg, 0.046 mmol) was dissolved in 1M HCl/EtOAc (240 μL) andstirred approximately 48 hours. The mixture was evaporated to dryness toprovide compound 11b as a light yellow paste and was coupled to compound4 (18 mg, 0.049 mmol) as described in Example 10, using NMM (20.3 μL,0.185 mmol) and HATU (21.1 mg, 0.056 mmol) in CH₂Cl₂. The crude materialwas purified by flash chromatography (eluent-hexane:Et₂O; 50:50) toprovide the dipeptide 11c as an oil (7.7 mg; 31%). By TLC, HPLC and NMRcomparison, dipeptide 11c, was found to be identical to the less polarcompound 10a obtained in Example 10, thus identifying the absolutestereochemistry of 10a as (1R,2R).

Example 12 Preparation of (1R, 2R)/(1S, 2R)1-Boc-amino-2-ethylcyclopropylcarboxylic acid: (12a)

The carbamate 9e from Example 9 (2.6 g, 7.88 mmol) was stirred for 40min in TFA at 0° C. The mixture was then concentrated and diluted withTHF (10 mL). An aqueous NaOH solution (700 mg, 17.5 mmol in 8.8 mL ofH2O ) was added followed by a THF (13 mL) solution of (Boc)₂O (2.06 g,9.44 mmol, 1.2 eq.). The reaction mixture was stirred overnight atRT(the pH was maintained at 8 by adding a 10% aqueous NaOH solution whenneeded), then diluted with H₂O, washed with Et₂O (3×) and acidified at0° C. with a 10% aq. citric acid solution. The aqueous layer wasextracted with EtOAc (3×) and successively washed with H₂O (2×) andbrine. After the usual treatment (MgSO₄, filtration and concentration)the desired Boc-protected amino acid (12a) (788 mg, 3.44 mmol, 44%yield) was isolated. ¹H NMR (CDCl₃) δ5.18 (bs, 1H), 1.64-1.58 (m, 2H),1.55-1.42 (m, 2H), 1.45 (s, 9H), 1.32-1.25 (m, 1H), 0.99 (t, 3H, J=7.3Hz).

Preparation of (1R, 2R)/(1S,2R)-1-Boc-amino-2-ethylcyclopropylcarboxylic acid methyl ester: (12b)

The Boc derivative 12a (0.30 g, 1.31 mmol) was dissolved in Et₂O (10 mL)and treated with freshly prepared diazomethane in Et₂O at 0° C. untilthe yellow color of a slight excess of diazomethane remained. Afterstirring for 20 min at RT the reaction mixture was concentrated todryness to give 12b as a clear colorless oil (0.32 g, 100%). ¹H NMR(CDCl₃) δ5.1 (bs, 1H), 3.71 (s, 3H), 1.62-1.57 (m, 2H), 1.55 (s, 9H),1.53-1.43 (m, 1H), 1.28-1.21 (m, 2H), 0.95 (t, J=7.3 Hz, 3H).

Example 13 Enzymatic resolution of methyl (1R, 2R)/(1S, 2R)Boc-1-amino-2-ethylcyclopropyl carboxylate

a) The enantiomeric mixture of (1S, 2R)/(1R, 2R)1-Boc-amino-2-ethylcarboxylic acid methyl ester of Example 10 (0.31 g,1.27 mmol) was dissolved in acetone (3 mL) and then diluted with water(7 mL) while being rapidly stirred. The pH of the solution was adjustedto 7.5 with 0.05M aqueous NaOH before Alcalase® [2.4L extract from NovoNordisk Industrials] (300 mg) was added. During incubation pH wasstabilized with NaOH and a pH stat was set up to monitor the addition ofthe NaOH solution. After 40 h the mixture was diluted with EtOAc and H₂O(with 5 mL sat. NaHCO₃) and the phases separated. The aqueous phase wasacidified with 10 10% aqueous HCl and extracted with EtOAc, dried(MgSO₄), filtered and concentrated to give acid 13a (48.5 mg). Theabsolute stereochemistry was determined using the correlation describedin Examples 10 and 11.

b) Treatment of an aliquot of acid 13a with diazomethane in Et₂O to givethe methyl ester followed by analysis by HPLC using a chiral column[Chiralcel® OD-H, 2.5% Isopropanol/hexane, isocratic] showed a 51:1ratio of the (S,R) isomer.

Example 14 Synthesis of (1R, 2S)/(1S, 2S) 1-amino-2-ethylcyclopropylcarboxylic acid

Starting from acid 9d described in Example 9:

c) To 9d (1.023 g, 4.77 mmol) in CH₃CN (25 mL) were successively addedDBU (860 μL, 5.75 mmol, 1.2 eq.) and allyl bromide (620 μL, 7.16 mmol,1.5 eq.). The reaction mixture was stirred for 4 h at RT and thenconcentrated. The residue was diluted with Et₂O and successively washedwith a 10% aq. citric acid solution (2×), H₂O, saturated aqueous NaHCO₃,H₂O (2×) and brine. After the usual treatment (MgSO₄, filtration andconcentration) the desired ester 14a was isolated (1.106 g, 3.35 mmol,91% yield) as a colorless oil. MS (FAB) 255 (MH⁺); ¹H NMR (CDCl₃)δ5.96-5.86 (m, 1H), 5.37-5.22 (m, 2H), 4.70-4.65 (m, 1H), 4.57-4.52 (m,1H), 1.87-1.79 (m, 1H), 1.47 (s, 9H), 1.45-1.40 (m, 1H), 1.33-1.24 (m,3H), 1.03 (t, J=7.3 Hz, 3H).

d) To ester 14a (1.106 g, 4.349 mmol) in dry CH₂Cl₂ (5 mL) at RT wasadded TFA (5 mL). The reaction mixture was stirred for 1.5 h and thenconcentrated to afford 14b (854 mg, 4.308 mmol, 99% yield). MS (FAB) 199(MH⁺); ¹H NMR (CDCl₃) δ5.99-5.79 (m, 1H), 5.40-5.30 (m, 2H), 4.71-4.62(m, 2H), 2.22-2.00 (m, 2H), 1.95-1.88 (m, 1H), 1.84-1.57 (m, 2H), 0.98(t, J=7.3 Hz, 3H).

e) To acid 14b (853 mg, 4.30 mmol) in dry benzene (14.8 mL) weresuccessively added Et₃N (684 μL, 4.91 mmol, 1.14 eq.) and DPPA (992 μL,4.60 mmol, 1.07 eq.). The reaction mixture was refluxed for 4.5 h then2-trimethylsilylethanol (1.23 mL, 8.58 mmol, 2.0 eq.) was added. Thereflux was maintained overnight then the reaction mixture was dilutedwith Et₂O and successively washed with a 10% aqueous citric acidsolution, water, saturated aq. NaHCO₃, water (2×) and brine. After theusual treatment (MgSO₄, filtration, concentration) the residue was flashchromatographed (5 cm, 10 to 15% AcOEt-hexane) to afford carbamate 14c(1.212 g, 3.866 mmol, 90% yield) as a pale yellow oil. MS (FAB) 314(MH⁺); ¹H NMR (CDCl₃) δ5.93-5.84 (m, 1H), 5.32-5.20 (m, 2H), 5.05 (bs,1H), 4.60-4.56 (m, 2H), 4.20-4.11 (m, 2H), 1.71-1.60 (m, 3H), 1.39-1.22(m, 1H), 1.03 (t, J=7.6 Hz, 3H), 0.96-0.86 (m, 1H), 0.04 (s, 9H).

f) To carbamate 14c (267 mg, 0.810 mmol) was added a 1.0 M TBAF solutionin THF (1.62 mL, 1.62 mmol, 2.0 eq.). The reaction mixture was stirredovernight at RT, refluxed for 30 min and then diluted with AcOEt. Thesolution was successively washed with water (2×) and brine. After theusual treatment (MgSO₄, filtration and concentration) the desired amine14d was isolated (122 mg, 0.721 mmol, 89% yield) as a pale yellowliquid. ¹H NMR (CDCl₃) δ5.94-5.86 (m, 1H), 5.31-5.22 (m, 2H), 4.58 (d,J=5.7 Hz, 2H), 1.75 (bs, 2H), 1.61-1.53 (m, 2H), 1.51-1.42 (m, 2H), 1.00(t, J=7.3 Hz, 3H), 0.70-0.62 (m, 1H).

Example 15 Synthesis of ethyl-(1R,2S)/(1S,2S)-1-amino-2-vinylcyclopropylcarboxylate

a) To a THF solution (180 mL) of potassium tert-butoxide (4.62 g, 41.17mmol, 1.1 eq.) at −78° C. was added commercially available imine 15a(10.0 g, 37.41 mmol) in THF (45 mL). The reaction mixture was warmed to0° C. and stirred at this temperature for 40 min. The mixture was thencooled back to −78° C. for the addition of 1,4-dibromobutene 15b (8.0 g,37.40 mmol) and then stirred at 0° C. for 1 h and cooled back to −78 °C. for the addition of potassium tert-butoxide (4.62 g, 41.17 mmol, 1.1eq.). The reaction mixture was finally stirred one more hour at 0° C.and concentrated to yield compound 15c.

b, c, d) 15c was taken up in Et₂O (265 mL) and treated with a 1N aq. HClsolution (106 mL). After 3.5 h at RT, the layers were separated and theaqueous layer was washed with Et₂O (2×) and basified with a saturatedaq. NaHCO₃ solution. The desired amine was extracted with Et₂O (3×) andthe combined organic extract was washed with brine. After the usualtreatment (MgSO₄, filtration and concentration) the residue was treatedwith a 4N HCl solution in dioxane (187 mL, 748 mmol). Afterconcentration, hydrochloride salt 15d was isolated as a brown solid(2.467 g, 12.87 mmol, 34% yield). ¹H NMR (CDCl₃) δ9.17 (bs, 3H),5.75-5.66 (m, 1H), 5.39 (d, J=17.2 Hz, 1H), 5.21 (d, J=10.2 Hz, 1H),4.35-4.21 (m, 2H), 2.77-2.70 (m, 1H), 2.05 (dd, J=6.4, 10.2 Hz, 1H),1.75 (dd, J=6.4, 8.3 Hz, 1H), 1.33 (t, J=7.0 Hz, 3H).

Example 16 Preparation of (1R,2S/1S,2S)-1-Boc-amino-2-vinylcyclopropylcarboxylic acid ethyl ester

The hydrochloride salt 15d (1.0 g, 5.2 mmol) and (Boc)₂O (1.2 g, 5.7mmol) were dissolved in THF (30 mL) and treated with DMAP (0.13 g, 1.04mmol, 0.2 equiv.) and diisopropylethylamine (2.8 mL, 15.6 mmol). Thereaction mixture was stirred 24 h before being diluted with EtOAc (40mL) and washed successively with sat. NaHCO₃ (aq), 5% aqueous HCl, andsat. brine. The organic phase was dried (MgSO₄), filtered andconcentrated to give after purification by flash chromatography (15%EtOAc/hexane), 16a (0.29 g, 23%). ¹H NMR (CDCl₃) δ5.80-5.72 (m, 1H),5.29-5.25 (dd, J=17.2, 17.2 Hz, 1H), 5.24-5.1 (bs, 1H), 5.10 (dd, J=9.2,9.2 Hz 1H), 4.22-4.13 (m, 2H), 2.15-2.04 (m, 1H), 1.85-1.73 (bs, 1H),1.55-1.5 (m, 1H), 1.49 (s, 9H), 1.26 (t, J=7.3 Hz, 3H).

Example 17 Enzymatic resolution of ethyl (1R,2S)/(1S,2S)1-amino-2-vinylcyclopropyl carboxylate

a) Racemic derivative 17a (0.29 g, 1.14 mmol) was dissolved in acetone(5 mL) and diluted with H₂O (10 mL). The pH was adjusted with 0.2Naqueous NaOH to 7.2 before Alcalase® was added (300 mg). To keep the pHconstant during incubation, a NaOH solution was added by a pH stattitrator over 9 days until the theoretical amount of base had beenadded. Following acid/base extraction as described in Example 13, theunhydrolyzed ester (0.15 g, 100%) and the hydrolyzed material (0.139 g,95%) were isolated. Analysis of the unhydrolyzed ester by HPLC using achiral column showed a ratio of 43:1 of the desired compound 17c thatwas assigned the (R,S) stereochemistry based on chemical correlation asdescribed in Examples 10 and 11.

Conditions for HPLC analysis: Chiralcel® OD-H (4.6 mm×25 cm), isocraticconditions using a mobile phase of 2.5% isopropanol/hexane.

Example 18 Resolution of (1R,2S)/(1S,2S) 1-amino-2-vinylcyclopropylcarboxylate by crystallization with dibenzoyl-D-tartaric acid

To a solution of crude racemic (1S,2S and 1R, 2S) ethyl1-amino-2-vinylcyclopropyl carboxylate [obtained fromN-(diphenylmethylene)glycine ethyl ester (25.0 g, 93.5 mol) as describedin Example 15] in EtOAc (800 mL) was added dibenzoyl-D-tartaric acid(33.5 g, 93.5 mol). The mixture was heated to reflux, left at RT for 15min then cooled to 0° C. A white solid was obtained after 30 min. Thesolid was filtered, washed with EtOAc (100 mL) and air-dried. The solidwas suspended in acetone (70 mL), sonicated and filtered (3×). The solidwas next recrystallized twice in hot acetone (crop A). The motherliquors were concentrated and the residue was recrystallized three timesin hot acetone (crop B). The two crops of the amorphous white solids ofdibenzoyl-D-tartaric acid salt were combined (5.53 g) and suspended in amixture of Et₂O (250 mL) and saturated NaHCO₃ solution (150 mL). Theorganic layer was washed with brine, dried (MgSO₄) and filtered. Thefiltrate was diluted with 1 N HCl/Et₂O (100 mL) and concentrated underreduced pressure. The oily residue was evaporated with CCl₄ to affordethyl 1 (R)-amino-2(S)-vinyl cyclopropanecarboxylate hydrochloride (940mg, 11% yield) as a white hygroscopic solid: [α]_(D) ²⁵+39.5° C. (c 1.14MeOH); [α]₃₆₅ ²⁵+88.5° C. (c 1.14 MeOH); ¹H NMR (DMSO-d₆) δ9.07 (broads, 2H), 5.64 (ddd, J=17.2, 10.4, 8.7 Hz, 1H), 5.36 (dd, J=17.2, 1.6 Hz,1H), 5.19 (dd, J=10.4, 1.6 Hz, 1H), 4.24-4.16 (m, 2H), 2.51-2.45 (m,peaks hindered by DMSO, 1H), 1.84 (dd, J=10.0, 6.0 Hz, 1H), 1.64 (dd,J=8.3, 6.0 Hz, 1H), 1.23 (t, J=7.1 Hz, 3H); MS (ESl) m/z 156 (MH)⁺; theenantiomeric purity was determined to be 91% ee by HPLC analysis(CHIRALPAK AS® column, Hex:i-PrOH) of the Boc derivative.

Example 19 Preparation of (1R,2S)/(1S, 2S)-1-amino-2-vinylcyclopropanecarboxylic acid methyl-ester hydrochloride (19f)

Preparation of imine 19b

Glycine ethyl ester hydrochloride 19a (1519.2 g, 10.88 mole, 1.0 equiv)was suspended in tert-butylmethyl ether (8 L). Benzaldehyde (1155 g,10.88 mole, 1 equiv) and anhydrous sodium sulfate (773 g, 5.44 mole, 05equiv) were added and the mixture cooled to 5 ° C. in an ice-water bath.Triethylamine (2275 mL, 16.32 mole, 1.5 equiv) was added dropwise over15 min (use 0.5 L of tert-butylmethyl ether for rinses) and the mixturestirred for 40 h at room temperature. The reaction was then quenched byaddition of ice-cold water (5 L) and the organic layer was separated.The aqueous phase was extracted with tert-butylmethyl ether (1 L) andthe combined organic phases washed with a mixture of saturated NaHCO₃(400 mL) and water (1.6 L), and then brine. The solution was dried overMgSO₄, concentrated under reduced pressure and the residual yellow oildried to constant weight under vacuum. Imine 19b was obtained as a thickyellow oil that solidifies at −20 ° C. (2001 g, 96% yield): ¹H NMR(CDCl₃, 400 MHz) δ8.30 (s, 1H), 7.79 (m, 2H), 7.48-7.39 (m, 3H), 4.40(d, J=1.3 Hz, 2H), 4.24 (q, J=7 Hz, 2H), 1.31 (t, J=7 Hz, 3H).

Preparation of racemic N-Boc-(1 R,2S)/(1S,2S)-1-amino-2-vinylcyclopropane carboxylic acid ethyl-esterhydrochloride 19e

Lithium tert-butoxide (4.203 g, 52.5 mmol, 2.1 equiv) was suspended indry toluene (60 mL). Imine 19b (5.020 g, 26.3 mmol, 1.05 equiv) anddibromide 19c (5.348 g, 25 mmol, 1 equiv) were dissolved in dry toluene(30 mL) and this solution added dropwise over 30 min to the stirredsolution of LiOtBu at room temperature. After completion, the deep redmixture was stirred for an additional 10 min and quenched by addition ofwater (50 mL) and tert-butylmethyl ether (TBME, 50 mL). The aqueousphase was separated and extracted a second time with TBME (50 mL). Theorganic phases were combined, 1 N HCl (60 mL) was added and the mixturestirred at room temperature for 2 h. The organic phase was separated andextracted with water (40 mL). The aqueous phases were then combined,saturated with salt (35 g) and TBME (50 mL) was added. The stirredmixture was then basified to pH 13-14 by careful addition of 10 N NaOH.The organic layer was separated and the aqueous phase extracted withTBME (2×50 mL). The organic extracts containing free amine 19d werecombined and ditertbutyldicarbonate (5.46 g, 25 mmol, 1 equiv) wasadded. After stirring overnight at room temperature, TLC showed someunreacted free amine. Additional ditertbutyldicarbonate (1.09 g, 5 mmol,0.2 equiv) was added and the mixture refluxed for 2 h, at which point,TLC analysis indicated complete conversion of 19d to carbamate 19e. Thesolution was cooled to room temperature, dried over MgSO₄ andconcentrated under reduced pressure. The residue was purified by flashchromatography using 10% then 20% EtOAc/hexane as eluent. Purified 19ewas obtained as a clear yellow oil which slowly solidifies under vacuum(4.014 g, 63% yield).

¹H NMR (CDCl₃, 400 MHz) δ5.77 (ddd, J=17, 10, 9 Hz, 1H), 5.28 (dd, J=17,1.5 Hz, 1H), 5.18 (broad s, 1H), 5.11 (dd J=10, 1.5 Hz, 1H), 4.24-4.09(m, 2H), 2.13 (q, J=8.5 Hz, 1H), 1.79 (broad m, 1H), 1.46 (m, 1H), 1.45(s, 9H), 1.26 (t, J=7 Hz, 3H).

Preparation of title compound 19f via trans-esterification of 19e

Ethyl ester 19e (10.807 g, 42.35 mmol) was dissolved in dry methanol (50mL) and a solution of sodium methoxide in MeOH (25% w/w, 9.7 mL, 42mmol, 1 equivalent) was added. The mixture was heated at 50° C. for 2 h,at which point TLC analysis indicated complete trans-esterification (19eR_(f)0.38, 19f R_(f)0.34 in 20% EtOAc/hexane). The reaction mixture wascooled to room temperature and acidified to pH 4 using 4N HCl indioxane. Precipitated NaCl was removed by filtration (usetert-butylmethyl ether for washings) and volatiles removed under reducedpressure. Tert-butylmethyl ether (100 mL) was added to the residue andsolids removed by filtration. Evaporation of the filtrate under reducedpressure and drying under vacuum gave pure methyl ester 19f (10.11 g,99% yield).

¹H NMR (CDCl₃, 400 MHz) δ5.75 (ddd, J=17, 10, 9 Hz, 1H), 5.28 (dd, J=17,1 Hz, 1H), 5.18 (broad s, 1H), 5.11 (ddd, J=10, 1.5, 0.5 Hz, 1H), 3.71(s, 3H), 2.14 (q, J=9 Hz, 1H), 1.79 (broad m, 1H), 1.50 (broad m, 1H),1.46 (s, 9H).

Example 20 Enzymatic resolution of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid methyl-ester hydrochloride

Preparation of N-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acidmethyl ester 20a

Racemic ester 19f (0.200 g, 0.83 mmol) was dissolved in acetone (3 mL)and water (7 mL) was added. 0.05 M NaOH (1 drop) was added to bring thepH of the solution to ˜8 and then Alcalase® 2.4L (Novo Nordisk Biochem,0.3 g in one mL of water) was added. The mixture was stirred vigorouslyat room temperature, maintaining the pH of the solution at 8 using anautomatic titrator. At beginning of day 4 and 5 of stirring at pH 8.3additional enzyme solution was added (2×0.3 g). After a total of 5 days,a total of 8.3 mL of 0.05 M NaOH was consumed. The reaction mixture wasdiluted with EtOAc and water and the organic phase separated. Afterwashing with brine, the organic extract was dried (MgSO₄) andconcentrated under vacuum. Compound 20a (0.059 g, 30% yield) wasobtained as a clear oil: ¹H NMR identical to that of compound 19f. HPLC(Chiralcel ODH, 4.6×250 mm, isocratic 1% EtOH in hexane, 0.8 mL/min flowrate): (1R,2S)-2 R_(t) 19.3 min (97%); (1S,2R)-2 R_(t) 17.0 min (3%).

Preparation of (1R,2S)-1-amino-2-vinylcyclopropane carboxylic acidmethyl ester hydrochloride 20b

Compound 20a (39.96 g, 165.7 mmol) was dissolved in dioxane (25 mL) andthe solution added dropwise with stirring to 4 N HCl in dioxane(Aldrich, 250 mL). After 45 min, TLC analysis indicated completedeprotection. Volatiles were removed under reduced pressure, and theresidue co-evaporated twice with MeOH (2×100 mL). Ether (300 mL) andMeOH (10 mL) were added to the brown, oily residue and the mixturestirred overnight at room temperature resulting in the precipitation ofa semi-solid. Additional MeOH (15 mL) was added and stirring continuedfor 6 h, at which point a yellowish solid was collected by filtration.The product was washed with 5% MeOH in ether (50 mL) and ether (2×50mL), and dried in vacuo to give compound 20b as a yellowish solid (22.60g, 76% yield). Filtrates (including washings) were evaporated in vacuumto give additional 20b as a brown oil (7.82 g, 26% yield). Bothfractions were pure enough for use in the synthesis of HCV proteaseinhibitors: [α]_(D) ²⁵+38.2° (c 1.0, MeOH).

¹H NMR (400 MHz, DMSO-d₆) δ9.15 (broad s, 3H), 5.65 (ddd, J=17, 10, 9Hz, 1H), 5.36 (dd, J=17, 1.5 Hz, 1H), 5.19 (dd, J=10, 1.5 Hz, 1H), 3.74(s, 3H), 2.50 (q, overlap with DMSO signal, J=9 Hz, 1H), 1.86 (dd, J=10,6 Hz, 1H), 1.64 (dd, J=8,6 Hz, 1H).

Example 21 Synthesis of 1-aminocyclobutyl carboxylic acid methyl ester

1,1-aminocyclobutanecarboxylic acid was prepared according to KavinDouglas; Ramaligam Kondareddiar; Woodard Ronald, Synth. Commun. (1985),15 (4), 267-72. The amino acid salt (21a) (1.00 g., 6.6 mmoles) wasstirred in dry methanol (40 ml) at −20° C. and mixture saturated withdry hydrogen chloride to yield (21b). Stirring of this mixture wascontinued for 4 h. The hot solution was filtered and filtrateconcentrated (Rotavap, 30° C. ) to leave a residue which upontrituration in ethyl ether afforded a white powder (0.907 g., 83% )after filtration and drying. ¹H NMR (400 MHz, D₂) δCH₃O (3H, s, 3.97ppm); CH₂ (2H, m, 2.70-2.77 ppm); CH₂ (2H, m, 2.45-2.53 ppm) and CH₂(2H, m, 2.14-2.29 ppm).

Tripeptides Example 22

General Procedure for Coupling Reactions Done on Solid Support

The synthesis was done on a parallel synthesizer model ACT396 fromAdvanced ChemTech® with the 96 well block. Typically, 24 peptides weresynthesized in parallel using standard solid-phase techniques. Thestarting (Fmoc-amino)cyclopropane (optionally substituted) carboxylicacid-Wang resin were prepared by the DCC/DMAP coupling method (Atherton,E; Scheppard, R. C. Solid Phase Peptide Synthesis, a Practical Approach;IRL Press: Oxford (1989); pp 131-148).

Each well was loaded with 100 mg of the starting resin (approximately0.05 mmol). The resins were washed successively with 1.5 mL portions ofNMP (1×) and DMF (3×). The Fmoc protecting group was removed bytreatment with 1.5 mL of a 25% v/v solution of piperidine in DMF for 20minutes. The resins were washed with 1.5 mL portions of DMF (4×), MeOH(3×) and DMF (3×). The coupling was done in DMF (350 μL), using 400 μL(0.2 mmol) of a 0.5M solution of Fmoc-amino acid/HOBt hydrate in DMF,400 μL (0.4 mmol) of a 1M solution of DIPEA in DMF and 400 μL (0.2 mmol)of a 0.5M solution of TBTU in DMF. After shaking for 1 hour, the wellswere drained, the resins were washed with 1.5 mL of DMF and the couplingwas repeated once more under the same conditions. The resins were thenwashed as described above and the cycle was repeated with the next aminoacid.

The capping groups were introduced in two ways:

1. In the form of a carboxylic acid using the protocol described above(for example acetic acid) or,

2. As an acylating agent such as an anhydride or an acid chloride. Thefollowing example illustrates the capping with succinic anhydride: Afterthe Fmoc deprotection and subsequent washing protocol, DMF was added(350 μL), followed by 400 μL each of a DMF solution of succinicanhydride (0.5 M, 0.2 mmol) and DIPEA (1.0 M, 0.4 mmol). The resins werestirred for 2 h and a recoupling step was performed. At the end of thesynthesis the resin was washed with 1.5 mL portions of DCM (3×), MeOH(3×), DCM (3×), and were dried under vacuum for 2 h. The cleavage fromthe resin and concomitant side chain deprotection was effected by theaddition of 1.5 mL of a mixture of TFA, H₂O, DTT and TIS (92.5: 2.5:2.5: 2.5). After shaking for 2.5 h, the resin was filtered and washedwith 1.5 mL of DCM. The filtrates were combined and concentrated byvacuum centrifugation. Each compound was purified by preparativereversed phase HPLC using a C18 column (22 mm by 500 mm). Theproduct-containing fractions were identified by MALDI-TOF massspectrometry, combined and lyophilized.

Example 23

General Procedure for Coupling Reactions Done in Solution {See Also R.Knorr et al., Tetrahedron Letters, (1989), 30, 1927.}

The reactants, i.e. a free amine (1 eq.) (or its hydrochloride salt) andthe free carboxylic acid (1 eq.) were dissolved in CH₂Cl₂, CH₃CN or DMF.Under a nitrogen atmosphere, four equivalents of N-methylmorpholine and1.05 equivalents of the coupling agent were added to the stirredsolution. After 20 min, one equivalent of the second reactant, i.e. afree carboxylic acid was added. (Practical and efficient couplingreagents for this purpose are(benzotriazol-1-yloxy)tris-(dimethylamino)phosphoniumhexafluorophosphate (HOBT) or preferably2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU) or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (HATU). The reaction was monitored by TLC. Aftercompletion of the reaction, the solvent was evaporated under reducedpressure. The residue was dissolved in EtOAc. The solution was washedsuccessively with 10% aqueous citric acid, saturated aqueous NaHCO₃ andbrine. The organic phase was dried (MgSO₄), filtered and concentratedunder reduced pressure. When the residue was purified, it was done byflash chromatography as defined above.

Example 24

Synthesis of Compound 304

a) The (R,R) isomer of Boc-Et-Acca-OMe 13c (0.12 g, 0.49 mmol) obtainedfrom enzymatic resolution (Example 13) was treated with 4N HCl/dioxane(45 min) before being concentrated in vacuo to give a white solid. Tothis HCl salt (ca. 0.49 mmol) was added TBTU (0.17 g, 0.54 mmol), theBoc-4(R)-(8-quinoline-methyloxy) proline 5 (from Example 5) (0.18 g,0.49 mmol) and DIPEA (0.3 mL, 1.7 mmol) in MeCN (10 mL). The mixture wasstirred at RT for 3.5 h before being concentrated in vacuo. Theresulting material was dissolved in EtOAc and washed sequentially withsaturated aqueous NaHCO₃ and brine. Dried (MgSO₄), filtered andconcentrated to give 24b a white solid (0.122 g, 50%).

b) 24b (0.12 g, 0.25 mmol) was treated at RT with 4N HCl/dioxane (30min) before being concentrated in vacuo. The resulting hydrochloridesalt (ca. 0.25 mmol) was treated with Boc-Chg-OH.H₂O (75 mg, 0.27 mmol),TBTU (87 mg, 0.27 mmol) in MeCN (10 mL) and finally at 0° C. with DIPEA(0.15 mL, 0.87 mmol). The residue was diluted with EtOAc, sequentiallywashed with saturated aqueous NaHCO₃, and brine, dried (MgSO₄), filteredand concentrated to give 24c as an off white solid (0.2 g). Thismaterial (0.14 g) was dissolved in DMSO and purified by preparative HPLCto give 24c as a white solid after lyophilization (35 mg, 33%). HPLC(98%); MS (FAB) m/z: 637.3 (MH⁺); HRMS calcd for C₃₅H₄₈N₄O₇ (MH⁺)637.36011: found 637.36250; ¹H-NMR (DMSO-d₆) shows a rotamer population,δ8.91 (2×d, J=4.1 and 4.1 Hz, 1H), 8.40-8.36 (m, 2H), 7.90 (d, J=7.6 Hz,1H), 7.77 (d, J=7.0 Hz, 1H), 7.6-7.54 (m, 2H), 6.80 (d, J=8.6 Hz, 1H),5.18 and 5.16 (2×s, 2H) 1H), 4.31 (t, J=8.3 Hz, 1H), 4.12 (d, J=11.44Hz, 1H), 4.03 (t, J=7.9 Hz, 1H), 3.78-3.72 (m, 1H), 3.56 (s, 3H),2.35-2.27 (m, 1H), 2.06-1.97 (m, 1H), 1.71-1.55 (m, 10H), 1.53-1.38 (m,2H), 1.26 (s, 9H), 1.18-1.06 (m, 2H), 1.02-0.93 (m, 2H), 0.89 (t J=7.3Hz, 3H).

Compound 304

c) To 24c (30 mg, ca. 0.047 mmol) was added MeOH (1 mL), THF (1 mL), andlithium hydroxide monohydrate (12 mg, 0.29 mmol) in H₂O (1 mL). Theclear solution was stirred rapidly for 48 h before being concentrated invacuo. The crude peptide was dissolved in DMSO and purified bypreparative HPLC to give compound 304 as a white solid afterlyophilization (21 mg, 72%). HPLC (99%); MS (FAB) m/z: (MH⁺) 623.3; HRMScalcd for C₃₄H₄₆N₄O₇ (MH⁺) 623.34448, found: 623.34630, ¹HNMR (DMSO-d₆)shows a rotamer population of 1:1, δ8.90 (2×d, J=4.1 Hz, 1H), 8.37 (d,J=8.3Hz, 1H), 8.26 (s, 1H), 7.89 (d, J=8.3Hz, 1H), 7.77 (d, J=6.7 Hz,1H), 7.6-7.53 (m, 2H), 6.88 and 6.79 (2×d, J=8.6 and 7.9 Hz, 1H), 5.17and 5.16 (2×s, 2H), 4.43-4.35 (bs, 1H), 4.29 (t, J=8.3 Hz, 1H),3.82-3.71 (m, 1H), 2.35-2.27 (m, 1H), 2.06-1.97 (m, 1H), 1.72-1.53 (m,10H), 1.52-1.44 (m, 2H), 1.37 and 1.29 (2×s, 9H), 1.18-1.05 (m, 3H),1.0-0.94 (m, 1H), 0.91 (t, J=7.3 Hz, 3H).

Example 25

Synthesis of Compound 301

a) Compound 25a (=12a) (282 mg, 1.23 mmol) was suspended in anhydrousCH₃CN (6 mL). DBU (221 μL, 1.48 mmol) and benzylbromide (161 μL, 1.35mmol) were added successively and the reaction mixture was stirredovernight at RT. The mixture was concentrated, the resulting oil wasdiluted with EtOAc and 10% aq. citric acid and successively washed with10% citric acid (2×), saturated aq. NaHCO₃ (2×), water (2×) and brine(1×). The EtOAc layer was dried (MgSO₄), filtered and evaporated todryness. The crude colorless oil was purified by flash chromatography(eluent-hexane: EtOAc; 95:5 to 90:10) to provide the benzylated product25b as a colorless oil (368 mg; 93%). MS (FAB) 318.2 MH⁻ 320.2 MH⁺ 342.2(M+Na)⁺ ¹H NMR (CDCl₃) δ7.37-7.28 (m, 5H), 5.22-5.10 (m, 1H), 5.19 (d,J=12 Hz, 1H) 5.16 (d, J=12 Hz, 1H), 1.60-1.40 (m, 4H), 1.39 (s, 9H),1.31-1.22 (m, 1H) 0.91 (t, J=7.5, 14.5 Hz, 3H).

b) Compound 25b (368 mg, 1.15 mmol) was treated with 4N HCl/dioxane (6mL) as described previously. The crude hydrochloride salt was coupled tocompound 4 (from Example 4) (470.8 mg, 1.27 mmol) with NMM (507μL, 4.61mmol) and HATU (instead of TBTU, 525.6 mg, 1.38 mmol) in CH₂Cl₂ (6 mL)as described in Example 22 to yield the crude racemic dipeptide as anorange oil. The crude material was purified by flash chromatography(eluent-hexane: Et₂O; 50:50) to provide the pure dipeptide 25c (the lesspolar eluting spot) as a white foam (223 mg; 68% of the theoreticalyield).

MS 571.4 MH⁻573.3 MH⁺595.3 (M+Na)⁺

¹H NMR (CDCl₃), ca. 1:1 mixture of rotamers, δ8.03 (b d, J=8 Hz, 1H),7.86 (b d, J=7.5 Hz, 1H), 7.82 (b d, J=6.5 Hz, 1H), 7.61 (b s, 0.5H),7.57-7.40(m, 4H), 7.31-7.21 (m, 5H), 6.48 (b s, 0.5H), 5.22-5.11 (m,1H), 5.08-4.81 (m, 3H), 4.41-3.74 (m, 3H), 3.49-3.18 (m, 1H), 2.76-1.90(m, 2H), 1.69-1.48 (m, 3H), 1.40 (s, 9H), 1.40-1.23 (m, 2H), 0.92 (t,J=7.5, 15 Hz, 3H).

c) The dipeptide 25c (170.1 mg, 0.297 mmol) was treated with 4NHCl/dioxane (2mL) as described previously. The crude hydrochloride saltwas coupled to Boc-Chg-OH (84.1 mg 0.327 mmol) with NMM (130.7 μL, 1.19mmol) and HATU (instead of TBTU, 135.5 mg; 0.356 mmol) in CH₂Cl₂ (2 mL)for 2.75 h at RT then worked up as described previously to provide thecrude tripeptide 25d as an ivory foam (ca. 211.4 mg; 100%).

MS (FAB) 712.5 MH⁺

Compound 301

d) The crude tripeptide 25d (ca. 15.4 mg, 0.022 mmol) was dissolved inabsolute ethanol (2 mL) and an estimated amount (tip of spatula) of both10% Pd/C catalyst and ammonium acetate were added. The mixture washydrogenated overnight under a hydrogen filled balloon at RT andatmospheric pressure. The reaction mixture was filtered through a 0.45μm Millex® filter, evaporated to dryness then diluted with EtOAc and 10%aqueous citric acid, and washed again with 10% aqueous citric acid (1×),water (2×) and brine (1×). The organic layer was dried (MgSO₄),filtered, evaporated to dryness and lyophilized to provide thetripeptide 301 as a white amorphous solid (11.0 mg; 82%).

MS (FAB) 622.5 MH+644.5 (M+Na)+ ¹H NMR (DMSO), ca. 1:4 mixture ofrotamers, δ8.54 & 8.27 (s, 1H), 8.06-7.99 (m, 1 H), 7.96-7.91 (m, 1H),7.87 (d, J=8 Hz, 1H), 7.57-7.42 (m, 4H), 6.81 (d, J=8 Hz, 1H), 4.99 (d,J=12 Hz, 1H), 4.88 (d, J=12 Hz, 1H), 4.46-4.19 (m, 2H), 4.17-4.02 (m,2H), 3.88-3.67 (m, 1H), 2.28-2.19 (m, 1H), 2.05-1.93 (m, 1H), 1.73-1.43(m, 8H), 1.32-1.07 (m, 6H), 1.28 (s, 9H), 1.03-0.85 (m, 2H), 0.91 (t,J=7.5, 15 Hz, 3H)

Example 26

Synthesis of Compound 306

a) The acid 26a (180 mg, 0.500 mmol) and the amine 15d (96 mg, 0.500mmole) were coupled using TBTU (192 mg, 0.600 mmol) and DIPEA (226 mg,1.75 mmol) in CH₂Cl₂ (10 mL) for 20 h. The reaction mixture wasconcentrated, taken up in ethyl acetate, washed twice with sat. NaHCO₃and once with brine. The organic layer was dried on MgSO₄, filtered andconcentrated to give 26c as a brown oil, used without purification inthe next step.

b, c) The crude compound 26c (ca. 0.500 mmol) was stirred for 30 min inHCl 4N/dioxane (4 mL) and concentrated to dryness. The solid was takenup in CH₂Cl₂ (10 mL) and DIPEA (226 mg, 1.75 mmol) was added followed byBoc-Chg-OH monohydrate (138 mg, 0.500 mmol) and TBTU (192 mg, 0.600mmol). The solution was stirred at RT for 5 h. The reaction mixture wasconcentrated, taken up in ethyl acetate, washed twice with sat. NaHCO₃and once with brine. The organic layer was dried on MgSO₄, filtered andconcentrated to give a brown oil, purified by flash chromatography togive 26d as a yellow oil, 204 mg, 64% over two couplings.

¹H NMR (CDCl₃) δ8.77-8.74 (m, 1 H), 8.14 (d, J=8 Hz, 1 H), 8.02 (d, J=9Hz, 1 H), 7.69 (dd, J=9, 7 Hz, 1 H), 7.52 (d, J=5 Hz, 1 H), 7.47 (dd,J=8, 7 Hz, 1 H), 6.78 (d, J=5 Hz, 1 H), 5.80-5.70 (m, 1 H), 5.35-5.27(m, 2 H), 5.14-5.07 (m, 2 H), 4.89-4.83 (m, 1 H), 4.39-4.32 (m, 1 H),4.30-4.24 (m, 1 H), 4.20-4.07 (m, 2 H), 4.00-3.92 (m, 1 H), 3.04-2.92(m, 1 H), 2.39-2.29 (m, 1 H), 2.16-2.04 (m, 1 H), (m, 1 H), 1.91-1.83(m, 1 H), 1.82-1.62 (m, 7 H), 1.45-1.35 (m, 9 H), 1.27-1.07 (m, 8 H).

d) 26d (136 mg, 0.214 mmole) was dissolved in THF (4 mL) and MeOH (2mL). An aqueous solution (2 mL) of LiOH hydrate (72 mg, 1.72 mmol) wasadded and the reaction mixture was stirred at RT for 20 h. The solutionwas concentrated and purified by preparative HPLC to give compound 306(the less polar isomer) as a white solid (25 mg).

compound 306: MS(FAB) 607.4 (MH+)

¹H NMR (DMSO-d₆) δ9.16 (d, J=6 Hz, 1 H), 8.55 (s, 1 H), 8.35 (d, J=8 Hz,1 H), 8.12 (d, J=9 Hz, 1 H), 8.05 (dd, J=8, 7 Hz, 1 H), 7.76 (dd, J=8, 7Hz, 1 H), 7.59 (d, J=6 Hz, 1 H), 7.02 (d, J=8 Hz, 1 H), 5.75-5.66 (m,2H), 5.19 (d, J=18 Hz, 1 H), 5.07 (d, J=10 Hz, 1 H), 4.55 (d, J=12 Hz, 1H), 4.43 (dd, J=10, 8 Hz, 1 H), 4.03 (d, J=10 Hz, 1 H), 3.87-3.83 (m, 1H), 2.66-2.59 (m, 1 H), 2.36-2.30 (m, 1 H), 1.98 (dd, J=18, 9 Hz, 1 H),1.75-1.56 (m, 8 H), 1.38-1.35 (m, 1 H), 1.25-1.22 (m,1 H), 1.09 (s, 9H), 1.12-0.95 (m, 3 H).

Example 27

Synthesis of Compound 307

c) A solution of the acid (8) from Example 8 (505 mg, 105 mmol) in 5 mLof dichloromethane was treated with TBTU (376 mg, 1.17 mmol). The HClsalt of the (R,S) vinyl AccaOEt (18) (from Example 18) (279 mg, 1.46mmol), in 7 mL of dichloromethane containing (0.60 mL, 5.46 mmol) ofN-methyl morpholine, was added to the previous solution of the activatedester. The resulting solution was stirred at RT overnight. The solventwas evaporated in vacuo. The residue diluted with ethyl acetate, waswashed twice with a saturated solution of sodium bicarbonate and oncewith brine. The organic layer was dried (MgSO₄) filtered and evaporatedin vacuo. The residue was chromatographed over silica gel (60:40 v/v,hexanes-ethyl acetate) to afford 173 mg (27%) of the dipeptide 27c.

d, e) A solution of the dipeptide 27c (70 mg, 0.114 mmol) in 3 mL ofhydrogen chloride 4.0 M solution in 1,4-dioxane was stirred at RT for 1h (a precipitated came out from the reaction after 10 min). The solventwas removed in vacuo. The amine hydrochloride salt 27d (0.114 mmol),diluted in 1.5 mL of acetonitrile, was neutralized by addition of 65 μL(0.591 mmol) of N-methyl morpholine. A solution of the Boc ChgOH.H₂O (39mg, 0.142 mmol) in 1.5 mL of acetonitrile was treated with TBTU (46 mg,0.143 mmol) and then added to the previous solution of the amine. Theresulting solution was stirred at RT (for 2 days). The solvent wasremoved in vacuo. The residue, diluted with ethyl acetate, was washedtwice with a saturated solution of sodium bicarbonate and once withbrine. The organic layer was dried (MgSO₄), filtered and evaporated invacuo. 86 mg (100%) of tripeptide 27e was obtained. This crude compoundwas used in the next reaction without further purification.

f) To a solution of tripeptide 27e (86 mg, 0.114 mmol) in 5 mL of amixture THF:H₂O (2.5:1) was added lithium hydroxide monohydrate (22 mg,0.524 mmol). An additional 0.25 mL of MeOH was added in order to get anhomogeneous solution. The resulting solution was stirred at RT overnightbefore the solvent was evaporated in vacuo. The residue was partitionedbetween water and EtOAc. The aqueous layer was acidified with 1M HCl andthen extracted twice with ethyl acetate. The desired compound has beenfound in the ethyl acetate coming from the first basic extraction. Thisorganic layer was dried (MgSO₄), filtered and evaporated in vacuo toafford 69 mg of the crude acid, which was purified by preparatory HPLC.The compound was dissolved in MeOH (4 mL) and injected onto anequilibrated Whatman Partisil 10-ODS-3 (2.2×50 cm) C18 reverse phasecolumn. (λ=230 nm, solvent A=0.06% TFA/H₂O, solvent B=0.06% TFA/CH₃CN).Purification program: 20% to 70% of solvent B in 60 min. Fractions wereanalyzed by analytical HPLC. Appropriate fractions were collected andlyophilized to provide 50 mg (60%) of the desired tripeptide 307 as awhite amorphous solid.

compound 307: ¹H NMR (DMSO-d₆) rotamers ˜2:8 δ8.86 (d, J=2.5 Hz, 1H),8.85 (s, 0.2H), 8.64 (s, 0.8H), 8.49 (dd, J=9.5, 3 Hz, 0.2H), 8.45 (dd,J=9.2 Hz, 0.8H), 8.39-8.33 (m, 2H), 8.20 (d, J=9.5 Hz, 0.2H), 8.18 (d,J=9.5 Hz, 0.8H), 7.81 (s, 0.2H), 7.78 (s, 0.8H), 7.64-7.56 (m, 3H), 6.87(d, J=8 Hz, 0.8H), 6.36 (d, J=9 Hz, 0.2H), 5.82-5.67 (m, 2H), 5.27-5.17(m, 1H), 5.09-5.03 (m, 1H), 4.73 (t, J=8 Hz, 0.2H), 4.55 (dd, J=10, 7.5Hz, 0.8H), 4.49-4.40 (m, 1H), 4.00-3.95 (m, 1H), 3.83 -3.76 (m, 1H),2.87-2.80 (m, 0.2H), 2.69-2.62 (m, 0.8H), 2.39-2.26 (m, 1H), 2.08-2.00(m, 1H), 1.75-1.41 (m, 7H), 1.37 (s, 1.8H), 1.32-1.27 (m, 1H), 1.17-0.82(m, 5H), 0.94 (s, 7.2H).

Example 28

Synthesis of Compound 311

Compound 311 was prepared using the process described in Example 24 butusing the appropriate building blocks.

Compound 310 ¹H NMR (DMSO-d₆) δ8.98 (d, J=6 Hz, 1 H), 8.52 (S, 1 H),8.24 (d, J=9 Hz, 1 H), 8.08 (d, J=2 Hz, 1 H), 7.63 (d, J=9 Hz, 1 H),7.37 (d, J=6 Hz, 1 H), 6.98 (d, J=8 Hz, 1 H), 5.75-5.66 (m, 1 H), 5.57(br s, 1 H), 5.24-5.19 (m, 1 H), 5.08-5.01 (m, 1 H), 4.57-4.40 (m, 2 H),4.00-3.96 (m, 1 H), 3.82 (dd, J=9, 8 Hz, 1 H), 2.59-2.54 (m, 1 H),2.32-2.26 (m, 1 H), 1.99 (dd, J=17, 9 Hz, 1 H), 1.74-1.55 (m, 8 H), 1.37(s, 1 H), 1.26-1.22 (m, 1 H), 1.14-1.08 (m, 9 H), 1.02-0.91 (m, 3 H).

Example 29

Synthesis of Compound 302

Compound 302 was prepared using the process described in Example 27 butusing the appropriate building blocks.

¹H NMR (DMSO-dr) δ8.34 (s, 1 H), 8.04-8.01 (m, 1 H), 7.94-7.92 (m, 1 H),7.87 (d, J=8 Hz, 1 H), 7.54-7.50 (m, 3 H), 7.45 (dd, J=17, 8 Hz, 1 H),7.22 (d, J=8 Hz, 1 H), 4.94 (dd, J=55, 12 Hz, 2 H), 4.34 (s, 1 H), 4.27(dd, J=8, 8 Hz, 1 H), 4.16 (d, J=11 Hz, 1 H), 4.07 (dd, J=8, 8 Hz, 1 H),3.72-3.65 (m, 2 H), 3.59-3.54 (m, 1 H), 2.24-2.18 (m, 1 H), 2.02-1.95(m, 2 H), 1.75-1.70 (m, 1 H), 1.53-1.44 (m, 2 H), 1.32-1.27 (m, 1 H),1.21-1.17 (m, 1 H), 0.96-0.85 (m, 10 H), 0.80-0.77 (m, 5 H), 0.62-0.57(m, 1 H).

Example 30

Synthesis of Compound 308

Compound 308 was prepared using the process described in Example 27 butusing the appropriate building blocks.

¹H NMR (DMSO-d₆) rotamers ≡2:8 δ8.77 (s, 0.2H), 8.45 (s, 0.8H), 8.13 (d,J=8.5 Hz, 0.8H), 8.03 (d, J=8.5 Hz, 0.2H), 7.89-7.83 (m, 1 H), 7.55-7.37(m, 4H), 7.05-6.59 (m, 1H), 6.95 (d, J=8 Hz, 0.8H), 6.26 (d, J=8.5 Hz,0.2H), 5.81-5.64 (m, 1H), 5.33-5.28 (m, 1H), 5.26-5.15 (m, 1H),5.08-5.02 (m, 1H), 4.60 (t, J=7.5 Hz, 0.2H), 4.38-4.27 (m, 1.8H),4.09-3.91 (m, 1.8H), 3.74 (dd, J=12.5, 4 Hz, 0.2H), 2.69-2.60 (m, 0.2H)2.50-2.40 (m, 1H), 2.36-2.28 (m, 0.2H), 2.23-2.14 (m, 0.8H), 2.05-1.97(m, 0.8H), 1.76-1.44 (m, 7H), 1.37 (s, 1.8H), 1.29 (s, 7.2H), 1.28-1.20(m, 1H), 1.16-0.88 (m, 5H).

Example 31

Synthesis of Compound 309

Compound 309 was prepared using the process described in Example 27 butusing the appropriate building blocks.

¹H NMR (DMSO-d₆) rotamers ≡2:8 δ8.75 (s, 0.2H), 8.50 (s, 0.8H),7.89-7.78 (m, 3H), 7.50-7.44 (m, 1H), 7.42-7.32 (m, 2H), 7.17-7.09 (m,0.8H) 7.08-7.03 (m, 0.2H), 6.79 (d, J=8.5 Hz, 0.8H), 6.33 (d, J=9 Hz,0.2H), 5.81-5.65 (m, 1H), 5.30-5.16 (m, 2H), 5.10-5.02 (m, 1H), 4.56 (t,J=7.5 Hz, 0.2H), 4.33 (t, J=8 Hz, 0.8H), 4.10-3.90 (m, 2.8H), 3.74-3.68(m, 0.2H), 2.45-2.37 (m, 1H), 2.34-2.17 (m, 1H), 2.05-1.97 (m, 1H),1.76-1.48 (m, 7H), 1.37 (s, 1.8H), 1.23 (s, 7.2H), 1.21-0.88 (m, 6H).

Example 32

Synthesis of Compound 305

Compound 305 was prepared using the process described in Example 27 butusing the appropriate building blocks.

¹H NMR (DMSO-d₆) rotamers (1:9) δ8.68 (s, 0.1H), 8.43 (s, 0.9H),8.04-8.00 (m, 1H), 7.95-7.91 (m, 1H), 7.87 (d, J=8.5 Hz, 1H), 7.57-7.49(m, 3H), 7.47-7.42 (m, 1H), 6.82 (d, J=8.5 Hz, 0.9H), 6.21 (d, J=8.5 Hz,0.1 H), 5.80-5.64 (m, 1H), 5.21 (dd, J=17, 2 Hz, 0.1H), 5.18 (dd, J=17,2 Hz, 0.9H), 5.06 (dd, J=10.5, 2 Hz, 1H), 5.02-4.85 (m, 2H), 4.43 (t,J=7.5 Hz, 0.1H), 4.34 (br s, 1H), 4.23 (t, J=8.5 Hz, 0.9H), 4.16-4.05(m, 1.8H), 3.89-3.82 (m, 0.2H), 3.74 (dd, J=11, 3.5 Hz, 0.9H), 3.53 (dd,J=12.5, 4 Hz, 0.1 H), 2.30-2.21 (m, 1H), 2.02-1.94 (m, 2H), 1.74-1.38(m, 7H), 1.36 (s, 0.9H), 1.28 (s, 8.1 H), 1.25-0.87 (m, 6H).

Example 33

Synthesis of Compound 303

Compound 303 was prepared using the process described in Example 27 butusing the appropriate building blocks.

¹H NMR (DMSO-d₆) δ8.29 (s, 1 H), 8.04-8.01 (m, 1 H), 7.94-7.92 (m, 1 H),7.87 (d, J=8 Hz, 1 H), 7.56-7.52 (m, 3 H), 7.46 (dd, J=8, 7 Hz, 1 H),7.19 (d, J=9 Hz, 1 H), 5.01 (d, J=12 Hz, 1 H), 4.86 (d, J=12 Hz, 1 H),4.34 (br. s, 1 H), 4.24 (t,J=8 Hz, 1 H), 4.18-4.09 (m, 2 H), 3.74-3.53(m, 3 H), 2.24-2.18 (m, 1 H), 2.04-1.95 (m, 1 H), 1.74-1.45 (m, 10 H),1.31-1.13 (m, 4 H), 0.96-0.86 (m, 7 H), 0.79-0.76 (m, 5 H).

Example 34

Synthesis of Compound 403

a) Coupling of P2 with P1

The methyl ester derivative of 7 (34a) (170 mg, 0.355 mmole) was stirredin 50% THF-methanol (4 ml) and aqueous LiOH (1M, 1 ml) at RT for 1 h.The solution was concentrated (Rotavap, 30° C.) and residue acidified topH 6 and solution lyophilized. The resulting powder was stirred in dryDMF (3 ml) in the presence of DIEA (0.4 ml) followed by the successiveaddition of 1,1-aminocyclobutylcarboxylic acid methyl esterhydrochloride (34b) (140 mg, 0.845 mmole) and TBTU (134 mg, 0.417mmole). After stirring for 18 h at RT, the mixture was purified by flashchromatography on silica gel (230-400 Mesh) using 1:2 ethylacetate-hexane to afford an orange oil (98 mg, 90% purity by HPLC).

b) Coupling of P1-P2 with P3

The dipeptide 34b (97 mg, 90%, 0.155 mmole) was stirred in 4NHCl-dioxane (5 ml) during 1 h at RT. The solution was then concentratedto dryness (Rotavap, high vacuum) to afford a beige solid. This materialwas stirred in dry DMF (2 ml) at RT in the presence of DIEA (0.4 ml)followed by addition of L-Boc-Tbg (80 mg, 0.35 mmole) and TBTU (112 mg,0.35 mmole). After stirring 2 days at RT, the solution was poured inethyl acetate to generate the free base using 5% aqueous potassiumcarbonate. The organic phase was worked up to give a yellow oilyresidue. The material was purified by flash chromatography on silica gelcolumn (230-400 Mesh) using 1:2 & 3:1 v/v ethyl acetate: hexane toafford 40 mg of an oil, homogeneous by HPLC.

The methyl ester (40 mg) was finally saponified in 1N potassiumhydroxide (2 ml) in methanol (4 ml) by stirring at RT during 3 h. Themixture was concentrated (Rotavap, 30° C.) and acidified to pH 4 with 2Nhydrochloric acid. This mixture was purified by preparative HPLC on C18column using a gradient of 0-50% aqueous acetonitrile (0.1% TFA) at 220nm The fractions were pooled, concentrated to half volume andlyophilized to afford 403 as a white fluffy solid (10 mg).

¹H NMR (400 MHz, DMSO-d₆) δ Mixture of rotamers: NH+ (1H, s, 8.6 ppm),CH (3H, m, 8.2 ppm), Ph (5H, broad s, 7.66 & 7.53 ppm), CH (1H, broad,7.22 ppm), NH (1H, d, J=7.6 Hz, 6.71 ppm), CHO (1H, broad s, 5.76 ppm),CH (2H, m, 4.58-4.49 ppm), CH (1H, m, 4.04 ppm), CH₃O (3H, s, 3.97 ppm),CH (1H, d, 3.86 ppm), CH (7H, very broad, 1.8-2.6 ppm), Boc group (9H,s, 1.25 ppm) and t-butyl group (9H, s, 0.97 ppm).

MS. showed M+H+at m/e 675 (100%).

HPLC peak 98% at 18.9 min.

Example 35

Synthesis of Compound 333 (Table 3)

A solution of m-anisidine (35a) (9.15 mL, 81.4 mmoles) anddimethylacetylene-dicarboxylate (35b) (10.0 mL, 81.3 mmoles) in 160 mLof methanol is heated under reflux for 2 h. The solvent is removed invacuo and the residue purified by a flash column chromatography (90:10hexanes-ethyl acetate). Compound 35c (17.0 g, 79% yield) is obtained asan orange oil.

¹H NMR (CDCl₃) δ9.62 (broad s, 1H), 7.17 (dd, J=7 and 8.5 Hz, 1H),6.66-6.62 (m, 1H), 6.49-6.45 (m, 2H), 5.38 (s, 1H), 3.77 (s, 3H), 3.74(s, 3H), 3.71 (s, 3H).

Diphenylether (50 mL) is heated in a sand bath up to an internaltemperature of ≈250°. Diester adduct (35c) (7.5 g, 28.3 mmoles),dissolved in 5 mL of diphenyl ether, is added within 2 min to theboiling solvent. The heating is maintained for 5 min and the reactionmixture is then allowed to cool down to room temperature. Rapidly abeige solid precipitated out. The solid is filtered and then trituratedwith methanol. To yield 4.1 g (62% yield) of the desired compound 35d.

¹H NMR (DMSO-d₆) δ7.97 (d, J=9 Hz, 1H), 7.40 (d, J=2 Hz, 1H), 6.96 (dd,J=9 and 2 Hz, 1H), 6.55 (s, 1H), 3.95 (s, 3H), 3.84 (s, 3H).

A solution of cis-4-hydroxy-L-proline derivative (35e) (1.71 g, 5.33mmoles), 4-hydroxyquinoline derivative (35f) (1.25 g, 5.36 mmoles) andtriphenylphosphine (2.80 g, 10.68 mmoles) in 75 mL of THF is cooled downto 0° for the addition drop to drop (≈1 h) of DEAD (1.70 mL, 10.80mmoles). The reaction mixture was then allowed to warm up slowly to roomtemperature and the stirring was continued overnight. The solvent isremoved in vacuo and the residue purified by a flash columnchromatography (70:30 ehtylacetate-hexanes). Compound 35g (0.7 g of purecompound 35 g, and 1.8 g of compound 35 g contaminated with ≈50% oftriphenylphosphate oxide) is obtained as a white solid.

¹H NMR (CDCl₃) rotamers (4:6) δ8.04 (d, J=9 Hz, 1H), 7.54 (d, J=2.5 Hz,1 H), 7.40-7.32 (m, 6H), 7.23 (dd, J=9 and 2.5 Hz, 1H), 5.33-5.13 (m,3H), 4.66 (t, J=7.5 Hz, 0.4 H), 4.54 (t, J=8 Hz, 0.6 H), 4.07 (s, 3H),3.94 (s, 3H), 4.04=3.80 (m, 2H), 2.78-2.65 (m, 1H), 2.47-2.34 (m, 1H),1.45 (s, 3.6H), 1.37 (s, 5.4H).

To proline benzyl ester derivative (35 g) (0.70 g, 1.31 mmoles) insolution in a mixture of methanol-ethyl acetate (10 mL-10 mL) is added100 mg of 10% Pd/C. The resulting suspension is stirred at roomtemperature under hydrogen atmosphere for 1½ h. The catalyst is thenfiltered on a Millex-HV Millipore (0.45 μm filter unit) and the solventsare evaporated in vacuo. Quantitative yield of the desired acid 35h(0.59 g) is obtained.

¹H NMR: (CDCl₃) rotamers 70:30 δ8.06 (d, J=9.5 Hz, 0.3 H), 8.01 (d, J=9Hz, 0.7 H), 7.56 (d, J=2 Hz, 1H), 7.44 (broad s, 0.7 H), 7.41 (broad s,0.3 H), 7.24 (dd, J=9 and 2.5 Hz, 1H), 5.31-5.25 (m, 1H), 4.67 (t, J=7.5Hz, 0.7 H), 4.55 (t, J=7.5 Hz, 0.3 H), 4.08 (s, 3H), 3.95 (s, 3H),4.04-3.80 (m, 2H), 2.83-2.72 (m, 1H), 2.71-2.47 (m, 1H), 1.46 (s, 9H).

The salt of the amine 35i (215 mg, 1.21 mmoles) in 7 mL of acetonitrileis treated with 0.95 mL of DIEA (5.45 mmoles). This solution is thenadded to a solution of acid 35h (590 mg, 1.32 mmoles) and TBTU (389 mg,1.21 mmoles) in 5 mL of CH₃CN the resulting solution is stirred at roomtemperature overnight. The solvent is removed in vacuo and the residueis diluted with ethylacetate and washed twice with a saturated solutionof sodium bicarbonate once with brine and dried over MgSO₄. The solventis removed in vacuo and the residue is purified by flash columnchromatography (75:25 AcOEt-hexanes) to afford 527 mg (70% yield) of thedesired dipeptide (35j).

¹H NMR: (CDCl₃) δ8.01 (d, J=9 Hz, 1H), 7.55 (d, J=2.5 Hz, 1H), 7.45 (s,1H), 7.22 (dd, J=9 and 2.5 Hz, 1H), 5.81-5.71 (m, 1H), 5.36-5.28 (m,2H), 5.18-5.12 (m, 1H), 4.61-4.45 (m, 1H), 4.07 (s, 3H), 3.94 (s, 3H),3.91-3.74 (m, 2H), 3.72 (s, 3H), 2.99-2.84 (m, 1H), 2.49-2.34 (m, 1H),2.20-2.08 (m, 1H), 1.97-1.84 (m, 1H), 1.58-1.52 (m, 1H), 1.44 (s, 9H).

The diester 35j (716 mg, 1.25 mmoles) in solution in a mixture ofTHF:MeoH (1.5 mL—1.5 mL) is cooled to 0° before being treated with anaqueous solution of NaOH 1M (1.25 mL, 1.25 mmoles). After 1 h ofstirring at 0°, 3 drops of glacial acetic acid are added to neutralizethe NaOH. The solvents are removed in vacuo and the compound is dried onthe pump for a few hours.

A solution of the acid 35k sodium salt (1.25 mmoles) and Et₃N (0.19 mL,1.36 mmoles) in 8 mL of THF is cooled to 0° and isobutyl chloroformate(0.18 mL, 1.39 mmoles) is added. After 40 min diazomethane (9 mL, 6.30mmoles) is added and the resulting solution is stirred at 0° for 30 minand at room temperature for 2 h. The solvents are removed in vacuo. Theresidue, diluted with ethyl acetate, is washed twice with a saturatedsolution of NaHCO₃ once with brine and dried over MgSO₄, the solvent isevaporated under vacuo and the residue is purified by flash columnchromatography (50:50 Hexanes/AcOEt) to afford 378 mg (52% yield) of theexpected diazoketone 35l.

¹H NMR: (CDCl₃) δ8.00 (d, J=9 Hz, 1H), 7.42 (s, 1H), 7.35 (d, J=2.5 Hz,1H), 7.20 (dd, J=9 and 2.5 Hz, 1H), 6.92 (s, 1H), 5.81-5.71 (m, 1H),5.35-5.28 (m, 3H), 5.17-5.13 (m, 1H), 4.61-4.40 (m, 1H), 3.97 (s, 3H),3.96-3.74 (m, 2H), 3.72 (s, 3H), 2.94-2.38 (m, 2H), 2.18-2.06 (m, 1H),1.98-1.84 (m, 1H), 1.57-1.52 (m, 1H), 1.42 (s, 9H).

To a cooled (0°) solution of the diazoketone 35l (0.37 g, 0.642 mmoles)in 15 mL of THF is added 0.25 mL of HBr 48%. The resulting yellowsolution is stirred at 0° for 1 h. The reaction mixture is partitionedbetween ethyl acetate and a saturated solution of NaHCO₃. The organicphase is washed one more time with NaHCO₃ and dried with NaSO₄. Afterevaporation of the solvents in vacuo, 0.36 g (90% yield) of theα-bromoketone 35m is isolated.

The α-bromoketone 35m (170 mg, 0.271 mmoles) in 10 mL of isopropanol istreated with 1-acetyl-2-thiourea (64 mg, 0.542 mmoles). The resultingsolution is heated at 75° for 1 h. The solvent is removed in vacuo. Theresidue is diluted with ethyl acetate and washed twice with a saturatedsolution of NaHCO₃, once with brine and dried with MgSO₄. Evaporation ofthe solvent in vacuo afforded 182 mg (>100%) of crude material 35n.

The dipeptide 35n (145 mg, 0.223 mmoles) is treated with 3 mL of a 4Msolution of HCl in dioxane. The resulting solution is stirred at roomtemperature for 1 h. The solvents are removed in vacuo and the residueis dried over the pump.

The salt of the amine 35o in 5 mL of CH₃CN is treated with 195 μL (1,12mmoles) of DIEA. This solution is then added to the solution of theBoc-tert-butylglycine (103 mg, 0.446 mmoles) and HATU (186 mg, 0.489mmoles) in 3 mL of CH₃CN. The reaction mixture is stirred at roomtemperature overnight. The CH₃CN is evaporated in vacuo. The residuediluted with ethyl acetate is washed twice with a saturated solution ofNaHCO₃, once with brine and dried with MgSO₄. After removal of thesolvent, 274 mg of the crude tripeptide 35p is obtained (>100%).

The tripeptide 35p (56 mg, 0.0733 mmoles), in 4 mL of a 4M solution ofHCl in dioxane, is stirred at room temperature for 2 h. The solvent isremoved in vacuo and the residue dried over the pump.

The salt of the amine obtained is dissolved in 4 mL of CH₂Cl₂ andtreated with 0.13 mL of DIEA (0.746 mmoles) followed by 26 mg oftriphosgene (0.0876 mmoles). After 3 h incubation,1,2,2-trimethylpropylamine (20 mg, 0.146 mmoles) is added (synthesizedas described in Moss N., Gauthier J., Ferland J. M., Feb. 1995, SynLett.(2), 142-144). The ice bath is removed and the reaction mixture isstirred at room temperature overnight. The CH₂Cl₂ is evaporated invacuo. The residue, diluted with ethyl acetate is washed twice with asaturated solution of NaHCO₃, once with brine and dried with MgSO₄ toafford 60 mg (≈100%) of the desired urea 35q.

A solution of methyl ester 35q (57 mg, 0.0721 mmoles) in a mixture ofTHF:H₂O (2.5 mL: 1 mL) is treated with solid LiOHoH₂O (25 mg, 0.595mmoles) and 1 mL of MeOH is added in order to clarify the solution.After stirring for 4 h at room temperature, the reaction is neutralizedby addition of a 1M solution of HCl. The solvents are removed in vacuoand the residue is purified by a preparative chromatography. Thecompound dissolved in 2.5 mL of MeOH, is injected into an equilibratedWhatman Partisil 10-ODS-3 (2.2×50 cm) C₁₈ reverse phase column.Purification program: Linear Gradient at 20 mL/nm, λ220 nm, inject at10% A up to 60% A in 60 min. A:0.06% TFA/CH₃CN; B:0.06%; TFA/H₂O.Fractions were analyzed by analytical HPLC. The product collected waslyophilized to provide 15 mg of compound 333 as an off white solid (27%yield).

¹H NMR: (DMSO-d₆) δ8.88 (s, 0.2H), 8.84 (d, J=4.5 Hz, 0.2H), 8.68 (d,J=8.5 Hz, 0.0H), 8.56 (s, 0.8H), 8.40-8.13 (m, 1,5H), 7.96 (d, J=9.0 Hz,0.2H), 7.72-7.44 (m, 2.4H), 7.35-7.09 (m, 1.2H), 6.98 (d, J=9 Hz, 0.2H),6.15 (d, J=9 Hz, 0.2H), 6.06 (d, J=9 Hz, 0.8H), 5.93 (d, J=9.5 Hz,0.24H), 5.86 (d, J=9 Hz, 0.8H), 5.79-5.67 (m, 1H), 5.69-5.44 (m, 1H),5.24-5.14 (m, 1H), 5.09-5.01 (m, 1H), 4.50-4.35 (m, 2H), 4.24 (d, J=9.0Hz, 0.2H), 4.20 (d, J=9.0 Hz, 0.8H), 4.06-3.98 (m, 2H), 3.95 (s, 3H),3.77-3.60 (m, 2H), 2.58-2.50 (m, 1H), 2.22 (s, 2.4H), 2.21 (s, 0.6H),2.02 (q, J−9 Hz, 1H), 1.56-1.38 (m, 1H), 1.28-1.22 (m, 1H), 0.97 (s,9H), 0.83 (d, J=6 Hz, 3H), 0.72 (s, 9H).

MS(FAB) 778.3 (m+H)⁺, 776.3 (M−H)⁻.

Example 36

Cloning, Expression and Purification of the Recombinant HCV NS3 ProteaseType 1b

Serum from an HCV-infected patient was obtained through an externalcollaboration (Bernard Willems MD, Hôpital St-Luc, Montréal, Canada andDr. Donald Murphy, Laboratoire de Santé Publique du Québec, Ste-Anne deBellevue, Canada). An engineered full-length cDNA template of the HCVgenome was constructed from DNA fragments obtained by reversetranscription-PCR (RT-PCR) of serum RNA and using specific primersselected on the basis of homology between other genotype 1b strains.From the determination of the entire genomic sequence, a genotype 1b wasassigned to the HCV isolate according to the classification of Simmondset al. (J. Clin. Microbiol., (1993), 31, p. 1493-1503). The amino acidsequence of the non-structural region, NS2-NS4B, was shown to be greaterthan 93% identical to HCV genotype 1b (BK, JK and 483 isolates) and 88%identical to HCV genotype 1a (HCV-1 isolate). A DNA fragment encodingthe polyprotein precursor (NS3/NS4A/NS4B/NS5A/NS5B) was generated by PCRand introduced into eukaryotic expression vectors. After transienttransfection, the polyprotein processing mediated by the HCV NS3protease was demonstrated by the presence of the mature NS3 proteinusing Western blot analysis. The mature NS3 protein was not observedwith expression of a polyprotein precursor containing the mutationS1165A, which inactivates the NS3 protease, confirming the functionalityof the HCV NS3 protease.

The DNA fragment encoding the recombinant HCV NS3 protease (amino acid1027 to 1206) was cloned in the pET11d bacterial expression vector. TheNS3 protease expression in E. coli BL21 (DE3)pLysS was induced byincubation with 1 mM IPTG for 3 h at 22° C. A typical fermentation (18L) yielded approximately 100 g of wet cell paste. The cells wereresuspended in lysis buffer (3.0 mL/g) consisting of 25 mM sodiumphosphate, pH 7.5, 10% glycerol (v/v), 1 mM EDTA, 0.01% NP-40 and storedat −80° C. Cells were thawed and homogenized following the addition of 5mM DTT. Magnesium chloride and DNase were then added to the homogenateat final concentrations of 20 mM and 20 μg/mL respectively. After a 25min incubation at 4° C., the homogenate was sonicated and centrifuged at15000×g for 30 min at 40° C. The pH of the supernatant was then adjustedto 6.5 using a 1M sodium phosphate solution.

An additional gel filtration chromatography step was added to the 2 steppurification procedure described in WO 95/22985 (incorporated herein byreference). Briefly, the supernatant from the bacterial extract wasloaded on a SP HiTrap column (Pharmacia) previously equilibrated at aflow rate of 2 mL/min in buffer A (50 mM sodium phosphate, pH 6.5, 10%glycerol, 1 mM EDTA, 5 mM DTT, 0.01% NP-40). The column was then washedwith buffer A containing 0.15 M NaCl and the protease eluted by applying10 column volumes of a linear 0.15 to 0.3 M NaCl gradient. NS3protease-containing fractions were pooled and diluted to a final NaClconcentration of 0.1 M. The enzyme was further purified on a HiTrapHeparin column (Pharmacia) equilibrated in buffer B (25 mM sodiumphosphate, pH 7.5, 10% glycerol, 5 mM DTT, 0.01% NP-40). The sample wasloaded at a flow rate of 3 mL/min. The column was then washed withbuffer B containing 0.15 M NaCl at a flow rate of 1.5 mL/min. Two stepwashes were performed in the presence of buffer B containing 0.3 or 1MNaCl. The protease was recovered in the 0.3M NaCl wash, diluted 3-foldwith buffer B, reapplied on the HiTrap Heparin column and eluted withbuffer B containing 0.4 M NaCl. Finally, the NS3 protease-containingfractions were applied on a Superdex 75 HiLoad 16/60 column (Pharmacia)equilibrated in buffer B containing 0.3 M NaCl. The purity of the HCVNS3 protease obtained from the pooled fractions was judged to be greaterthan 95% by SDS-PAGE followed by densitometry analysis.

The enzyme was stored at −80° C. and was thawed on ice and diluted justprior to use.

Example 37

Recombinant HCV NS3 Protease/NS4A Cofactor Peptide Radiometric Assay

The enzyme was cloned, expressed and prepared according to the protocoldescribed in Example 36. The enzyme was stored at −80° C., thawed on iceand diluted just prior to use in the assay buffer containing the NS4Acofactor peptide. The substrate used for the NS3 protease/NS4A cofactorpeptide radiometric assay, DDIVPC-SMSYTW, is cleaved between thecysteine and the serine residues by the enzyme. The sequenceDDIVPC-SMSYTW corresponds to the NS5A/NS5B natural cleavage site inwhich the cysteine residue in P2 has been substituted for a proline. Thepeptide substrate DDIVPC-SMSYTW and the tracerbiotin-DDIVPC-SMS[¹²⁵I-Y]TW are incubated with the recombinant NS3protease and the NS4A peptide cofactor KKGSWIVGRIILSGRK (molar ratioenzyme: cofactor 1:100) in the absence or presence of inhibitors. Theseparation of substrate from products is performed by addingavidin-coated agarose beads to the assay mixture followed by filtration.The amount of SMS[¹²⁵I-Y]TW product found in the filtrate allows for thecalculation of the percentage of substrate conversion and of thepercentage of inhibition.

A. Reagents

Tris and Tris-HCl (UltraPure) were obtained from Gibco-BRL. Glycerol(UltraPure), MES and BSA were purchased from Sigma. TCEP was obtainedfrom Pierce, DMSO from Aldrich and NaOH from Anachemia.

Assay buffer: 50 mM Tris HCl, pH 7.5, 30% (w/v) glycerol, 1 mg/mL BSA, 1mM TCEP (TCEP added just prior to use from a 1 M stock solution inwater).

Substrate: DDIVPCSMSYTW, 25 μM final concentration (from a 2 mM stocksolution in DMSO stored at −20° C. to avoid oxidation).

Tracer: reduced mono iodinated substrate biotin DDIVPC SMS[¹²⁵I Y]TW (˜1nM final concentration).

HCV NS3 protease type 1b, 25 nM final concentration (from a stocksolution in 50 mM sodium phosphate, pH 7.5, 10% glycerol, 300 mM NaCl, 5mM DTT, 0.01% NP-40).

NS4A Cofactor peptide: KKGSVVIVGRIILSGRK, 2.5 μM final concentration(from a 2 mM stock solution in DMSO stored at −20° C).

B. Protocol

The assay was performed in a 96-well polystyrene plate from Costar. Eachwell contained:

20 μL substrate/tracer in assay buffer;

10 μL ± inhibitor in 20% DMSO/assay buffer;

10 μL NS3 protease 1b/NS4 cofactor peptide (molar ratio 1:100).

Blank (no inhibitor and no enzyme) and control (no inhibitor) were alsoprepared on the same assay plate.

The enzymatic reaction was initiated by the addition of the enzyme/NS4Apeptide solution and the assay mixture was incubated for 40 min at 23°C. under gentle agitation. Ten (10) μL of 0.5N NaOH were added and 10 μL1 M MES, pH 5.8 were added to quench the enzymatic reaction.

Twenty (20) μL of avidin-coated agarose beads (purchased from Pierce)were added in a Millipore MADP N65 filtration plate. The quenched assaymixture was transferred to the filtration plate, and incubated for 60min at 23° C. under gentle agitation.

The plates were filtered using a Millipore MultiScreen Vacuum ManifoldFiltration apparatus, and 40 μL of the filtrate was transferred in anopaque 96-well plate containing 60 μL of scintillation fluid per well.

The filtrates were counted on a Packard TopCount instrument using a¹²⁵I-liquid protocol for 1 minute.

The % inhibition was calculated with the following equation:

100−[(counts_(inh)−counts_(blank))/(counts_(ctl)−counts_(blank))×100]

A non-linear curve fit with the Hill model was applied to theinhibition-concentration data, and the 50% effective concentration(IC₅₀) was calculated by the use of SAS software (Statistical SoftwareSystem; SAS Institute, Inc. Cary, N.C.).

Example 38

Full-length NS3-NS4A Heterodimer Protein Assay

The NS2-NS5B-3′non coding region was cloned by RT-PCR into the pCR®3vector (Invitrogen) using RNA extracted from the serum of an HCVgenotype 1b infected individual (provided by Dr. Bernard Willems,Hôpital St-Luc, Montréal, Québec, Canada). The NS3-NS4A DNA region wasthen subcloned by PCR into the pFastBac™ HTa baculovirus expressionvector (Gibco/BRL). The vector sequence includes a region encoding a28-residue N-terminal sequence which contains a hexahistidine tag. TheBac-to-Bac™ baculovirus expression system (Gibco/BRL) was used toproduce the recombinant baculovirus. The full length mature NS3 and NS4Aheterodimer protein (His-NS3-NS4AFL) was expressed by infecting 10⁶ Sf21cells/mL with the recombinant baculovirus at a multiplicity of infectionof 0.1-0.2 at 27° C. The infected culture was harvested 48 to 64 h laterby centrifugation at 4° C. The cell pellet was homogenized in 5 mMNaPO₄, pH 7.5, 40% glycerol (w/v), 2 mM β-mercaptoethanol, in presenceof a cocktail of protease inhibitors. His-NS3-NS4AFL was then extractedfrom the cell lysate with 1.5% NP-40, 0.5% Triton X-100, 0.5M NaCl, anda DNase treatment. After ultracentrifugation, the soluble extract wasdiluted 4-fold and bound on a Pharmacia Hi-Trap Ni-chelating column. TheHis-NS3-NS4AFL was eluted in a >90% pure form (as judged by SDS-PAGE),using a 50 to 400 mM imidazole gradient. The His-NS3-NS4AFL was storedat −80° C. in 50 mM sodium phosphate, pH 7.5, 10% (w/v) glycerol, 0.5 MNaCl, 0.25 M imidazole, 0.1% NP-40. It was thawed on ice and dilutedjust prior to use. The protease activity of His-NS3-NS4AFL was assayedin 50 mM Tris-HCl, pH 8.0, 0.25 M sodium citrate, 0.01% (w/v)n-dodecyl-β-D-maltoside, 1 mM TCEP. Five (5) μM of the internallyquenched substrate anthranilyl-DDIVPAbu[C(O)—O—]-AMY(3-NO₂)TW-OH inpresence of various concentrations of inhibitor were incubated with 1.5nM of His-NS3-NS4AFL for 45 min at 23° C. The final DMSO concentrationdid not exceed 5.25%. The reaction was terminated with the addition of1M MES, pH 5.8. Fluorescence of the N-terminal product was monitored ona Perkin-Elmer LS-50B fluorometer equipped with a 96-well plate reader(excitation wavelength: 325 nm; emission wavelength: 423 nm).

The % inhibition was calculated with the following equation:

100−[(counts_(inh)−counts_(blank))/(counts_(ctl)−counts_(blank))×100]

A non-linear curve fit with the Hill model was applied to theinhibition-concentration data, and the 50% effective concentration(IC₅₀) was calculated by the use of SAS software (Statistical SoftwareSystem; SAS Institute, Inc. Cary, N.C.).

Example 39

NS3 Protease Cell-based Assay

This assay was done with Huh-7 cells, a human cell line derived from ahepatoma, co-transfected with 2 DNA constructs:

one expressing a polyprotein comprising the HCV non-structural proteinsfused to tTA in the following order: NS3-NS4A-NS4B-NS5A-tTA (calledNS3);

the other expressing the reporter protein, secreted alkalinephosphatase, under the control of tTA (called SEAP).

The polyprotein must be cleaved by the NS3 protease for the matureproteins to be released. Upon release of the mature proteins, it isbelieved that the viral proteins will form a complex at the membrane ofthe endoplasmic reticulum while tTA will migrate to the nucleus andtransactivate the SEAP gene. Therefore, reduction of NS3 proteolyticactivity should lead to reduction of mature tTA levels and concomitantdecrease in SEAP activity.

To control for other effects of the compounds, a parallel transfectionwas done where a construct expressing tTA alone (called tTA) wasco-transfected with the SEAP construct such that SEAP activity isindependent of NS3 proteolytic activity. Protocol of the assay: Huh-7cells, grown in CHO-SFMII+10% FCS (fetal calf serum), wereco-transfected with either NS3 and SEAP or tTA and SEAP, using theFuGene protocol (Boehringer Mannheim). After 5 h at 37°, the cells werewashed, trypsinized and plated (at 80 000 cells/well) in 96-well platescontaining a range of concentrations of the compounds to be tested.After a 24-h incubation period, an aliquot of the medium was drawn andthe SEAP activity in this aliquot was measured with the Phospha-Lightkit (Tropix).

Analysis of the percent inhibition of SEAP activity with respect tocompound concentration was performed with the SAS software to obtain theEC₅₀.

The toxicity of the compound (TC₅₀) was then assessed using the MTTassay as follows:

20 μL of a MTT solution (5 mg/ml medium) was added per well andincubated at 37° for 4 hrs;

the medium was removed and 50 μl of 0.01N HCl+10% Triton X-100 wasadded; after shaking at RT for at least 1 hr, the OD of each well wasread at 595 nm wavelength.

The TC₅₀ was calculated in the same way as the EC₅₀.

Example 40

Specificity Assays

The specificity of the compounds was determined against a variety ofserine proteases: human leukocyte elastase, porcine pancreatic elastaseand bovine pancreatic α-chymotrypsin and one cysteine protease: humanliver cathepsin B. In all cases a 96-well plate format protocol using acolorimetric p-nitroaniline (pNA) substrate specific for each enzyme wasused. Each assay included a 1 h enzyme-inhibitor pre-incubation at 30°C. followed by addition of substrate and hydrolysis to ≈30% conversionas measured on a UV Thermomax® microplate reader. Substrateconcentrations were kept as low as possible compared to K_(M) to reducesubstrate competition. Compound concentrations varied from 300 to 0.06μM depending on their potency.

The final conditions for each assay were as follows:

50 mM Tris-HCl pH 8, 0.5 M Na₂SO₄, 50 mM NaCl, 0.1 mM EDTA, 3% DMSO,0.01% Tween-20 with;

[100 μM Succ-AAPF-pNA and 250 pM α-chymotrypsin], [133 μM Succ-AAV-pNAand 8 nM porcine elastase], [133 μM Succ-AAV-pNA and 8 nM leukocyteelastase]; or

[100 mM NaHPO₄ pH 6, 0.1 mM EDTA, 3% DMSO, 1 mM TCEP, 0.01% Tween-20, 30μM Z-FR-pNA and 5 nM cathepsin B (the stock enzyme was activated inbuffer containing 20 mM TCEP before use)].

A representative example is summarized below for porcine pancreaticelastase: In a polystyrene flat-bottom 96-well plate were added using aBiomek liquid handler (Beckman):

40 μL of assay buffer (50 mM Tris-HCl pH 8, 50 mM NaCl, 0.1 mM EDTA);

20 μL of enzyme solution (50 mM Tris-HCl pH 8, 50 mM NaCl, 0.1 mM EDTA,0.02% Tween-20, 40 nM porcine pancreatic elastase); and

20 μL of inhibitor solution (50 mM Tris-HCl, pH 8, 50 mM NaCl, 0.1 mMEDTA, 0.02% Tween-20, 1.5 mM-0.3 μM inhibitor, 15% v/v DMSO).

After 60 min pre-incubation at 30° C., 20 μL of substrate solution (50mM Tris-HCl, pH 8, 0.5 M Na₂SO₄, 50 mM NaCl, 0.1 mM EDTA, 665 μMSucc-AAA-pNA) were added to each well and the reaction was furtherincubated at 30° C. for 60 min after which time the absorbance was readon the UV Thermomax® plate reader. Rows of wells were allocated forcontrols (no inhibitor) and for blanks (no inhibitor and no enzyme).

The sequential 2-fold dilutions of the inhibitor solution were performedon a separate plate by the liquid handler using 50 mM Tris-HCl pH 8, 50mM NaCl, 0.1 mM EDTA, 0.02% Tween-20, 15% DMSO. All other specificityassays were performed in a similar fashion.

The percentage of inhibition was calculated using the formula:

[1−((UVinh−UVblank)/(UVctl−UVblank))]×100

A non-linear curve fit with the Hill model was applied to theinhibition-concentration data, and the 50% effective concentration(IC₅₀) was calculated by the use of SAS software (Statistical SoftwareSystem; SAS Institute, Inc., Cary, N.C.).

Tables of Compounds

The following tables list compounds representative of the invention.Compounds of the invention were assayed either in one or both of theassays of Examples 37 and 38 and were found to be active with IC₅₀ below50 μM.

Activity in cells and specificity:

Representative compounds of the invention were also tested in thesurrogate cell-based assay of Example 39, and in one or several assaysof Example 40. For example, compound 601 from Table 6 was found to havean IC₅₀ of 50 nM in the assay of Example 37 and 3 nM in the assay ofExample 38. The EC₅₀ as determined by the assay of Example 39 is 8.2 μM.In the specificity assays of Example 40, the same compound was found tohave the following activity: HLE >75 μM; PPE>75 μM; α-Chym.>75 μM; Cat.B>75 μM. These results indicate that this family of compounds is highlyspecific for the NS3 protease and at least certain members of thisfamily are active in a surrogate cell-based assay.

The following abbreviations are used within the present tables: MS: Massspectrometric data; Ac: acetyl; Bn: benzyl; Boc: tert-butyloxycarbonyl;cHex: cyclohexyl; Chg: cyclohexylglycine (2-amino-2-cyclohexyl-aceticacid); iPr: isopropyl; O-Bn: benzyloxy; Ph: phenyl: t-Bu: tert-butyl;Tbg: tert-butylglycine; 1- or 2-Np: 1- or 2-naphthyl; 1- or 2-NpCH₂O: 1,or 2-naphthylmethoxy.

TABLE 1

Tab 1 IC₅₀ Cpd# B R₃ R₂ MS (μM) 101 Boc cHex —O—CH₂—1-naphthyl 594 43102

cHex —O—CH₂—1-naphthyl 632 45 103

cHex —O—CH₂—1-naphthyl 642 42 104

cHex —O—CH₂—1-naphthyl 728 29.5 105

cHex —O—CH₂—1-naphthyl 619 47 106 Boc cHex

702 2.8 107

cHex —O—CH₂—1-naphthyl 720 M + Na⁺ 34 108 Boc iPr

662 8.9 109 acetyl cHex

644 6.3 110 Boc i-Pr

575.1 9.7 111 Boc t-Bu

661.3 0.475

TABLE 2

Table 2 R₁ anti IC₅₀ Cpd# B R₃ R₂ to carboxy MS (μM) 201 Boc cyclohexyl—O—CH₂—1-naphthyl ethyl 622 15 (one isomer) 202 Boc cyclohexyl—O—CH₂—1-naphthyl ethyl 622 40 (other isomer) 203 Boc t-Bu

vinyl 1R, 2R 687.5 0.082

TABLE 3

Table 3 R₁ syn to IC₅₀ Cpd # B R₃ R₂ carboxyl MS (μM) 301 Boc cHex—O—CH₂—1-naphthyl ethyl 622 7.7 302

iPr —O—CH₂—1-naphthyl ethyl 582 12.5 303

cHex —O—CH₂—1-naphthyl ethyl 622 11 304 Boc cHex

ethyl 623 32 305 Boc cHex —O—CH₂—1-naphthyl vinyl 620 3.2 306 Boc cHex

vinyl 607 0.8 307 Boc cHex

vinyl 728 0.27 308 Boc cHex

vinyl 606 1.6 309 Boc cHex

vinyl 606 5 310 Boc cHex

vinyl 607 2.5 311 Boc cHex

vinyl 641 0.56 312 Boc cHex

vinyl 607 8.5 313 Boc cHex

vinyl 621 2.5 314 Boc cHex

vinyl 683 0.14 315 Boc cHex

vinyl 698 0.66 316 Acetyl cHex

vinyl 625 1.9 317 Boc cHex

vinyl 740 0.32 318 CF₃—C(O)— i-Pr

vinyl 639.3 0.88 319

cHex

vinyl 732.3 1.2 320

cHex

vinyl 704.3 0.65 321 Boc t-Bu

vinyl 658.7 0.19 322 Boc t-Bu

vinyl 717.6 1.95 323 Boc t-Bu

672.4 0.64 324 Boc t-Bu

vinyl 727.5 0.05 325 Boc t-Bu

701.4 0.153 326 Boc t-Bu

vinyl 708.3 0.32 327

t-Bu

vinyl 610.3 0.045 328 Boc t-Bu

vinyl 615.3 3.2 329 Boc t-Bu

vinyl 685.3 0.36 330 Boc t-Bu

vinyl 627.5 6 331

t-Bu

vinyl 656.5 0.071 332 Boc t-Bu

ethyl 689.3 0.13 333

t-Bu

vinyl 778.3 0.003 334

t-Bu

vinyl 764.4 0.007

TABLE 4

Table 4 IC₅₀ Cpd # B R₃ R₂ R₁ (SM) 401 Boc i-Pr

H 589.1 5.8 402 Boc t-Bu

H 603.6 7.9 403 Boc t-Bu

H 675.4 0.132 404 Boc t-Bu

3-(═CH₂) 687.1 0.6 405 Boc t-Bu

2-vinyl 702.3 0.220 406 Boc t-Bu

2-Et 703.3 0.4

TABLE 5

Table 5 IC₅₀ Cpd# R₃ MS (μM) 501 t-Bu 581.3 0.4 502 H 539.2 6.2 503

625.3 0.79 504

582.6 2.6 505

583.2 0.79 506

659.2 1.3 507

670.2 0.98 508

703.3 3.1 509

581.3 0.377 510

581.2 0.255 511

637.2 2.1

TABLE 6

Table 6 IC₅₀ Cpd# R₃ R_(21A) R_(21B) MS (μM) 601 i-Pr Ph 7-OMe 673.30.05 602 t-Bu Ph 8-OMe, 717.2 0.041 7-OMe 603 i-Pr Ph 7-ethyl 671.20.195 604 t-Bu — 7-OMe 611.2 0.073 605 t-Bu Ph 7-O-iPr 715.3 0.195 606t-Bu — 7-Cl 615.2 0.48 607 iPr — 7-Cl 601.2 0.45 608 CH₂-iPr — 7-Cl615.3 1.45 609 t-Bu

— 680.2 1.7 610 t-Bu Cl — 613.3 0.25 611 t-Bu Ph 7-N(Me)₂ 700.5 0.035612 t-Bu

— 666.4 0.278 613 t-Bu

— 650.4 1.0 614 t-Bu

— 664.5 2.2 615 t-Bu — 7-N(Me)₂ 624.5 0.16 616 t-Bu

— 678.4 (M-H)⁺ 0.087 617 t-Bu

— 664.5 0.345 618 t-Bu

— 638.5 2.3 619 t-Bu

— 700.5 3.0 620 t-Bu

— 679.5 0.72 621 t-Bu

— 678.3 0.058 622 t-Bu

— 625.4 0.16 623 t-Bu MeO— — 611.3 0.20 624 t-Bu (Me)₂N— — 624.4 1.30625 t-Bu Ph 7-S(Me) 703.4 0.16 626 t-Bu Ph 7-Br 737.3 0.24 627 t-Bu Ph7-F 675.3 0.33 628 t-Bu

7-N(ME)₂ 764.2 0.011 629 t-Bu

7-N(Me)₂ 764.3 0.02 630 t-Bu

7--N(Et)₂ 792.3 0.043

TABLE 7

Table 7 IC₅₀ Cpd # R₃ R_(21A) MS (μM) 701 t-Bu

691.3 0.028 702 t-Bu

713.4 0.10 703 t-Bu

655.3 0.047 704 t-Bu

728.4 0.24 705 t-Bu

696.4 0.13 706 t-Bu

693.3 0.032 707 t-Bu

694.3 0.023 708 t-Bu Ph—N(Me)— 716.4 0.15 709 t-Bu

709.2 0.021 710 t-Bu HOOC— 655.3 0.685 711 t-Bu

708.2 0.016 712 t-Bu (Me)₂N— 654.3 0.10 713 t-Bu

692.3 (M-H)⁻ 0.026 714 t-Bu

722.3 0.012 715 t-Bu

688.3 0.031 716 t-Bu

688.3 0.079 717 t-Bu

723.3 0.028 718 t-Bu NH₂ 626.3 0.16 719 t-Bu

751.2 0.018 720 t-Bu

733.4 0.03 721 t-Bu

724.1 0.045 722 t-Bu

737.3 0.048 723 t-Bu

751.4 0.047 724 t-Bu

708.4 0.075 725 t-Bu

689.4 0.046 726 t-Bu i-Pr 653.3 0.25 727 t-Bu

688.3 0.07 728 t-Bu

786.1 0.022 729 t-Bu

689.3 0.2 730 t-Bu

669.2 0.042 731 t-Bu

669.2 0.031 732 t-Bu

791.0 0.02 733 t-Bu

765.3 0.028 734 t-Bu

671.3 0.044 735 t-Bu

683.3 0.058 736 t-Bu t-Bu 667.4 0.25 737 t-Bu CHex 693.4 0.11

TABLE 8

Table 8 IC₅₀ Cpd # B R₃ R₂₂ MS (μM) 801

t-Bu — 686.7 0.006 802

t-Bu — 727.7 0.024 803

t-Bu — 685.7 0.12 804

t-Bu — 711.7 0.032 805 Ac t-Bu — 629.6 0.083 806

t-Bu — 725.7 0.036 807

t-Bu — 672.4 0.01 808

t-Bu — 712.4 0.008 809

i-Pr — 649.3 0.071 810

t-Bu — 749.3 0.45 811 Boc t-Bu 4-Cl 721.3 0.04 812

t-Bu — 706.2 0.013 813

t-Bu — 702.2 0.02 814 Boc t-Bu 2-Cl 721.3 0.13 815 Boc t-Bu 3-Cl 721.30.16 816

t-Bu — 658.3 0.032 817

t-Bu — 720.2 0.017 818

t-Bu — 728.3 0.019 819

i-Pr — 762.3 0.32 820

i-Pr — 732.2 0.063 821

i-Pr — 679.1 0.12 822

i-Pr — 663.3 0.05 823 Boc t-Bu 2-OMe 717.2 0.107 824 Boc t-Bu 3-OMe719.2 0.07 825 Boc t-Bu 4-OMe 719.2 0.024 826

i-Pr — 663.3 0.78 827

t-Bu — 673.2 0.27 828

i-Pr — 691.3 0.10 829

t-Bu — 734.3 0.057 830

t-Bu — 645.3 0.111 831

t-Bu — 701.3 0.015 832

t-Bu — 801.3 0.11 833

t-Bu — 715.2 0.015 834

i-Pr — 663.3 0.074 835

t-Bu — 702.5 0.007 836

i-Pr — 694.4 0.13 837

i-Pr — 683.3 0.098 838

i-Pr — 679.1 0.094 839

i-Pr — 674.5 0.10 840

i-Pr — 667.4 0.085 841 Boc t-Bu 2-Me 701.5 0.24 842 Boc t-Bu 3-Me 701.50.073 843 Boc t-Bu 4-Me 701.5 0.053 844

t-Bu 4-OMe 716.6 0.006 845

i-Pr — 706.9 0.18 846

i-Pr — 693.4 0.104 847 Boc cHex — 713.4 0.037 848 Boc

— 687.5 0.093 849 Boc

— 701.5 0.110 850 Boc

— 731.5 0.063 851 Boc

— 689.5 0.12 852 Boc

— 689.5 0.05 853 Boc

— 765.5 0.17 854

i-Pr — 723.4 (M − H)⁺ 0.37 855

i-Pr — 693.3 0.075 856

i-Pr — 688.3 0.11 857

t-Bu — 716.4 0.011 858

t-Bu — 700.4 0.205 859

i-Pr — 655.4 0.83 860

i-Pr — 759.3 0.24 861

i-Pr — 688.3 0.17 862

i-Pr — 685.3 0.23 863

i-Pr — 699.4 0.30 864

i-Pr — 667.3 0.45 865

t-Bu — 701.4 0.02 866

t-Bu — 702.4 0.20 867

t-Bu — 701.3 0.051 868

t-Bu — 713.3 0.03 869

t-Bu — 699.4 0.014 870

t-Bu — 700.4 0.009 871

t-Bu — 714.3 0.011 872

t-Bu — 714.4 0.005 873

t-Bu — 714.3 0.019

TABLE 9

Table 9 IC₅₀ Cpd # B MS (μM) 901 Boc 685.3 0.025 902

825.4 0.042 903

769.3 0.005 904

707.3 0.095 905

685.2 0.029 906

728.2 0.014 907

717.2 0.025 908

691.2 0.072 909

727.2 0.036 910

715.3 0.056 911

721.3 0.039 912

733.2 0.034 913

713.3 0.030 914

805.3 0.031 915

692.2 0.026 916

680.3 0.3

TABLE 10

Table 10 IC₅₀ Cpd # B-X- R₃ Z R_(21B) MS (μM) 1001 Ph—N(Me)— i-Pr O H663.3 0.31 1002 Boc-NH— t-Bu S OMe 703.4 0.32 1003

i-Pr O — 663.3 0.31

What is claimed is:
 1. The racemates, diastereoisomers or opticalisomers of a compound of formula (I):

wherein B is H, a C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl; Het or (loweralkyl)-Het, all of which optionally substituted with C₁₋₆ alkyl; C₁₋₆alkoxy; C₁₋₆ alkanoyl; hydroxy; hydroxyalkyl; halo; haloalkyl; nitro;cyano; cyanoalkyl; amino optionally substituted with C₁₋₆ alkyl; amido;or (lower alkyl)amide; or B is an acyl derivative of formula R₄—C(O)—; acarboxyl derivative of formula R₄—O—C(O)—; an amide derivative offormula R₄—N(R₅)—C(O)—; a thioamide derivative of formulaR₄—N(R₅)—C(S)—; or a sulfonyl derivative of formula R₄—SO₂ wherein R₄ is(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amino optionally mono-or di-substituted with C₁₋₆alkyl, amido, or (lower alkyl) amide; (ii) C₃₋₇ cycloalkyl, C₃₋₇cycloalkoxy, or C₄₋₁₀ alkylcycloalkyl, all optionally substituted withhydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, amino optionally mono-ordi-substituted with C₁₋₆ alkyl, amido, or (lower alkyl) amide; (iii)amino optionally mono- or di-substituted with C₁₋₆ alkyl; amido; or(lower alkyl)amide; (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionallysubstituted with C₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, oramino optionally mono-or di-substituted with C₁₋₆ alkyl; or (v) Het or(lower alkyl)-Het, both optionally substituted with C₁₋₆ alkyl, hydroxy,amido, (lower alkyl) amide, or amino optionally mono-or di-substitutedwith C₁₋₆ alkyl; R₅ is H or C₁₋₆ alkyl; with the proviso that when B isa carboxyl derivative, an amide derivative or a thioamide derivative, R₄is not a cycloalkoxy; Y is H or C₁₋₆ alkyl; R³ is C₁₋₈ alkyl, C₃₋₇cycloalkyl, or C₄₋₁₀ alkylcycloalkyl, all optionally substituted withhydroxy, C₁₋₆ alkoxy, C₁₋₆ thioalkyl, amido, (lower alkyl)amido, C₆ orC₁₀ aryl, or C₇₋₁₆ aralkyl; R² is CH₂—R₂₀, NH—R₂₀, O—R₂₀ or S—R₂₀,wherein R₂₀ is pyridinyl, quinolyl, (lower alkyl)-pyridinyl or (loweralkyl)-quinolyl, each optionally mono-, di- or tri-substituted with R₂₁,wherein each R₂₁ is independently C₁₋₆ alkyl; C₁₋₆ alkoxy; lowerthioalkyl; sulfonyl; NO₂; OH; SH; halo; haloalkyl; amino optionallymono-or di-substituted with C₁₋₆ alkyl, C₆ or C₁₀ aryl, C₇₋₁₄ aralkyl,Het or (lower alkyl)-Het; amido optionally mono-substituted with C₁₋₆alkyl, C₆ or C₁₀ aryl, C₇₋₁₄ aralkyl, Het or (lower alkyl)-Het;carboxyl; carboxy(lower alkyl); C₆ or C₁₀ aryl, C₇₋₁₄ aralkyl or Het,said aryl, aralkyl or Het being optionally substituted with R₂₂; whereinR₂₂ is C₁₋₆ alkyl; C₃₋₇ cycloalkyl; C₁₋₆ alkoxy; amino optionally mono-or di-substituted with C₁₋₆ alkyl; sulfonyl; (lower alkyl)sulfonyl; NO₂;OH; SH; halo; haloalkyl; carboxyl; amide; (lower alkyl)amide; or Hetoptionally substituted with C₁₋₆ alkyl; R¹ is H; C₁₋₆ alkyl, C₃₋₇cycloalkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl, all optionally substitutedwith halogen; or a pharmaceutically acceptable salt or ester thereof;wherein at least one “Het” group is present in the compound of formula(I) and “Het” is defined as a six- or seven-membered saturated orunsaturated aromatic or heterocycle containing from one to fourheteroatoms selected from nitrogen, oxygen and sulfur, wherein saidheterocycle is optionally fused to a benzene ring.
 2. A compound offormula I according to claim 1, wherein B is a C₆ or C₁₀ aryl or C₇₋₁₆aralkyl, all optionally substituted with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano,cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substitutedwith C₁₋₆ alkyl; or B is Het or (lower alkyl)-Het, all optionallysubstituted with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl, hydroxy,hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (loweralkyl)amido, or amino optionally substituted with C₁₋₆ alkyl.
 3. Acompound of formula I according to claim 1, wherein B is R₄—SO₂ whereinR₄ is C₁₋₆ alkyl; amido; (lower alkyl)amide; C₆ or C₁₀ aryl, C₇₋₁₄aralkyl or Het, all optionally substituted with C₁₋₆ alkyl.
 4. Acompound of formula I according to claim 1, wherein B is an acylderivative of formula R₄—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyl optionallysubstituted with carboxyl, hydroxy or C₁₋₆ alkoxy, amido, (loweralkyl)amide, or amino optionally mono-or di-substituted with C₁₋₆ alkyl;(ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, both optionallysubstituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, or amino optionally mono-or di-substituted with C₁₋₆ alkyl;(iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substituted withC₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionallysubstituted with C₁₋₆ alkyl; (v) Het or (lower alkyl)-Het, bothoptionally substituted with C₁₋₆ alkyl, hydroxy, amino optionallysubstituted with C₁₋₆ alkyl, amido, (lower alkyl)amide, or aminooptionally substituted with C₁₋₆ alkyl.
 5. A compound of formula Iaccording to claim 1, wherein B is a carboxyl derivative of formulaR₄—O—C(O)—, wherein R₄ is (i) C₁₋₁₀ alkyl optionally substituted withcarboxyl, C₁₋₆ alkanoyl, hydroxy, C₁₋₆ alkoxy, amino optionally mono-ordi-substituted with C₁₋₆ alkyl, amido or (lower alkyl)amide; (ii) C₃₋₇cycloalkyl, C₄₋₁₀ alkylcycloalkyl, all optionally substituted withcarboxyl, (C₁₋₆ alkoxy)carbonyl, amino optionally mono- ordi-substituted with C₁₋₆ alkyl, amido or (lower alkyl)amide; (iv) C₆ orC₁₀ aryl or C₇₋₁₆ aralkyl optionally substituted with C₁₋₆ alkyl,hydroxy, amido, (lower alkyl)amido, or amino optionally mono-ordi-substituted with C₁₋₆ alkyl; or (v) Het or (lower alkyl)-Het, bothoptionally substituted with C₁₋₆ alkyl, hydroxy, amino optionallymono-or di-substituted with C₁₋₆ alkyl, amido or (lower alkyl)amido. 6.A compound of formula I according to claim 1, wherein B is an amidederivative of formula R₄—N(R₅)—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyloptionally substituted with carboxyl, C₁₋₆ alkanoyl, hydroxy, C₁₋₆alkoxy, amido, (lower alkyl)amido, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl; (ii) C₃₋₇ cycloalkyl or C₄₋₁₀alkylcycloalkyl, all optionally substituted with carboxyl, (C₁₋₆alkoxy)carbonyl, amido, (lower alkyl)amido, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl; (iii) amino optionally mono- ordi-substituted with C₁₋₃ alkyl; (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl,all optionally substituted with C₁₋₆ alkyl, hydroxy, amido, (loweralkyl)amide, or amino optionally substituted with C₁₋₆ alkyl; or (v) Hetor (lower alkyl)-Het, both optionally substituted with C₁₋₆ alkyl,hydroxy, amino optionally substituted with C₁₋₆ alkyl, amido or (loweralkyl)amide; and R₅ is H or methyl.
 7. A compound of formula I accordingto claim 1, wherein B is a thioamide derivative of formula R₄—NH—C(S)—;wherein R₄ is (i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆alkanoyl or C₁₋₆ alkoxy; (ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl,all optionally substituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, aminoor amido.
 8. A compound of formula I according to claim 2, wherein B isa C₆ or C₁₀ aryl optionally substituted with C₁₋₆ alkyl, C₁₋₆ alkoxy,C₁₋₆ alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano,cyanoalkyl, amido, (lower alkyl)amide, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl.
 9. A compound of formula I according toclaim 2, wherein B is Het optionally substituted with C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ alkanoyl, hydroxy, halo, amido, (lower alkyl)amide, oramino optionally mono- or di-substituted with C₁₋₆ alkyl.
 10. A compoundof formula I according to claim 4, wherein B is an acyl derivative offormula R₄—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyl optionally substitutedwith carboxyl, hydroxy or C₁₋₆ alkoxy; or (ii) C₃₋₇ cycloalkyl or C₄₋₁₀alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl,(C₁₋₆ alkoxy)carbonyl, or (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, alloptionally substituted with C₁₋₆ alkyl, hydroxy, or (v) Het optionallysubstituted with C₁₋₆ alkyl, hydroxy, amido or amino.
 11. A compound offormula I according to claim 5, wherein B is a carboxyl derivative offormula R₄—O—C(O)—, wherein R₄ is (i) C₁₋₁₀ alkyl optionally substitutedwith carboxyl, C₁₋₆ alkanoyl, hydroxy, C₁₋₆ alkoxy or amido, (loweralkyl)amide, amino optionally mono- or di-substituted with C₁₋₆ alkyl;(ii) C₃₋₇ cycloalkyl, C₄₋₁₀ alkylcycloalkyl, all optionally substitutedwith carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (lower alkyl)amide, aminooptionally mono- or di-substituted with C₁₋₆ alkyl, or (iv) C₆ or C₁₀aryl or C₇₋₁₆ aralkyl, all optionally substituted with C₁₋₆ alkyl,hydroxy, amino optionally substituted with C₁₋₆ alkyl; or (v) Het or(lower alkyl)-Het, both optionally substituted with C₁₋₆ alkyl, hydroxy,amido, or amino optionally mono-substituted with C₁₋₆ alkyl.
 12. Acompound of formula I according to claim 6, wherein B is an amidederivative of formula R₄—N(R₅)—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyloptionally substituted with carboxyl, C₁₋₆ alkanoyl, hydroxy, C₁₋₆alkoxy, amido, (lower alkyl)amide, amino optionally mono- ordi-substituted with C₁₋₆ alkyl; (ii) C₃₋₇ cycloalkyl or C₄₋₁₀alkylcycloalkyl, all optionally substituted with carboxyl, (C₁₋₆alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- ordi-substituted with C₁₋₆ alkyl; (iii) amino optionally mono- ordi-substituted with C₁₋₃ alkyl, or (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl,all optionally substituted with C₁₋₆ alkyl, hydroxy, amino or amidooptionally substituted with C₁₋₆ alkyl; or (v) Het optionallysubstituted with C₁₋₆ alkyl, hydroxy, amino or amido, and R₅ is H.
 13. Acompound of formula I according to claim 7, wherein B is a thioamidederivative of formula R₄—NH—C(S)—; wherein R₄ is (i) C₁₋₁₀ alkyl; or(ii) C₃₋₇ cycloalkyl.
 14. A compound of formula I according to claim 12,wherein B is an amide derivative of formula R₄—NH—C(O)— wherein R₄ is(i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy amido, (lower alkyl)amide, amino optionally mono-or di-substituted with C₁₋₆ alkyl; (ii) C₃₋₇ cycloalkyl or C₄₋₁₀alkylcycloalkyl, all optionally substituted with carboxyl, (C₁₋₆alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- ordi-substituted with C₁₋₆ alkyl; (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyloptionally substituted with C₁₋₆ alkyl, hydroxy, amino or amido.
 15. Acompound of formula I according to claim 1, wherein B is


16. A compound of formula I according to claim 1, wherein Y is H ormethyl.
 17. A compound of formula I according to claim 16, wherein Y isH.
 18. A compound of formula I according to claim 1, wherein R³ is C₁₋₈alkyl, C₃₋₇ cycloalkyl, or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with hydroxy, C₁₋₆ alkoxy, C₁₋₆ thioalkyl, acetamido, C₆ orC₁₀ aryl, or C₇₋₁₆ aralkyl.
 19. A compound of formula I according toclaim 18, wherein R³ is the side chain of Tbg, IIe, Val, Chg or:


20. A compound of formula I according to claim 19, wherein R³ is theside chain of Tbg, Chg or Val.
 21. A compound of formula I according toclaim 1, wherein R² is S—R₂₀ or O—R₂₀ wherein R₂₀ is a pyridinyl,quinolyl, —CH₂-pyridinyl or —CH₂-quinolyl, all optionally mono-, di-ortri-substituted with R₂₁, wherein R₂₁ is C₁₋₆ alkyl; C₁₋₆ alkoxy; lowerthioalkyl; amino or amido optionally mono- or di-substituted with C₁₋₆alkyl, C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl, Het or (lower alkyl)-Het; NO₂; OH;halo; trifluoromethyl; carboxyl; C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl, or Het,said aryl, aralkyl or Het being optionally substituted with R₂₂, whereinR₂₂ is C₁₋₆ alkyl; C₃₋₇ cycloalkyl; C₁₋₆alkoxy; amino; mono- ordi-(lower alkyl)amino; (lower alkyl)amide; sulfonylalkyl; NO₂; OH; halo;trifluoromethyl; carboxyl or Het.
 22. A compound of formula I accordingto claim 21, wherein R₂₁ is C₁₋₆ alkyl; C₁₋₆ alkoxy; amino; di(loweralkyl)amino; (lower alkyl)amide; C₆ or C₁₀ aryl, or Het, said aryl orHet being optionally substituted with R₂₂, wherein R₂₂ is C₁₋₆ alkyl;C₃₋₇ cycloalkyl; C₁₋₆ alkoxy; amino; mono- or di(lower alkyl)amino;amido; (lower alkyl)amide; halo; trifluoromethyl or Het.
 23. A compoundof formula I according to claim 22, wherein R₂₂ is C₁₋₆ alkyl; C₁₋₆alkoxy; halo; amino optionally mono- or di-substituted with lower alkyl;amido; (lower alkyl)amide; or Het.
 24. A compound of formula I accordingto claim 23, wherein R₂₂ is methyl; ethyl; isopropyl; tert-butyl;methoxy; chloro; amino optionally mono- or di-substituted with loweralkyl; amido, (lower alkyl)amide; or (lower alkyl) 2-thiazole.
 25. Acompound of formula I according to claim 21, wherein R² is


26. A compound of formula I according to claim 21, wherein R² isquinolinoxy unsubstituted, mono- or di-substituted with R₂₁ as definedin claim
 21. 27. A compound of formula I according to claim 26, whereinR² is selected from the group consisting of:


28. A compound of formula I according to claim 26, wherein R² is:

wherein R_(21A) is C₁₋₆ alkyl; C₁₋₆ alkoxy; lower thioalkyl; halo; aminooptionally mono-substituted with C₁₋₆ alkyl; or C₆, C₁₀ aryl, C₇₋₁₆aralkyl, or Het, said aryl, aralkyl or Het optionally substituted withR₂₂ wherein R₂₂ is C₁₋₆ alkyl, C₁₋₆ alkoxy, amido, (lower alkyl)amide,amino optionally mono- or di-substituted with C₁₋₆ alkyl, or Het; andR_(21B) is C₁₋₆ alkyl, C₁₋₆ alkoxy, amino, di(lower alkyl)amino, (loweralkyl)amide, NO₂, OH, halo, trifluoromethyl, or carboxyl.
 29. A compoundof formula I according to claim 28, wherein R_(21A) is C₆, C₁₀ aryl orHet, all optionally substituted with R₂₂ as defined in claim
 28. 30. Acompound of formula I according to claim 21, wherein R² is:

wherein R_(22A) is C₁₋₆ alkyl; C₁₋₆ alkoxy; or halo; and R_(21B) is C₁₋₆alkyl, C₁₋₆ alkoxy, amino, di(lower alkyl)amino, (lower alkyl)amide,NO₂, OH, halo, trifluoromethyl, or carboxyl.
 31. A compound of formula Iaccording to claim 30, wherein R_(21B) is C₁₋₆ alkoxy, or di(loweralkyl)amino.
 32. A compound of formula I according to claim 30, whereinR_(21B) is methoxy.
 33. A compound of formula I according to claim 1,wherein R¹ is H, C₁₋₃ alkyl, C₃₋₅ cycloalkyl, or C₂₋₄ alkenyl, alloptionally substituted with halo.
 34. A compound of formula I accordingto claim 33, wherein P1 is:

and R¹ is ethyl, vinyl, cyclopropyl, 1 or 2-bromoethyl or 1 or2-bromovinyl.
 35. A compound of formula I according to claim 34, whereinR¹ is vinyl.
 36. A compound of formula I according to claim 34, whereinR¹ at carbon 2 is orientated syn to the carbonyl at position 1,represented by the radical:


37. A compound of formula I according to claim 34, wherein R¹ atposition 2 is orientated anti to the carbonyl at position 1, representedby the radical:


38. A compound of formula I according to claim 34, wherein carbon 1 hasthe R configuration:


39. An optical isomer of a compound of formula I according to claim 38,wherein said R¹ substituent and the carbonyl in a syn orientation in thefollowing absolute configuration:


40. A compound of formula I according to claim 39, wherein R¹ is ethyl,hence the asymmetric carbon atoms at positions 1 and 2 have the R,Rconfiguration.
 41. A compound of formula I according to claim 39,wherein R¹ is vinyl, hence the asymmetric carbon atoms at positions 1and 2 have the R,S configuration.
 42. A compound of formula I accordingto claim 1, wherein B is a C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, alloptionally substituted with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl,hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido,(lower alkyl)amido, or amino optionally substituted with C₁₋₆ alkyl; orHet or (lower alkyl)-Het, all optionally substituted with C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl,nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionallysubstituted with C₁₋₆ alkyl,. or B is R₄—SO₂ wherein R₄ is preferablyamido; (lower alkyl)amide; C₆ or C₁₀ aryl, C₇₋₁₄ aralkyl or Het, alloptionally substituted with C₁₋₆ alkyl, or B is an acyl derivative offormula R₄—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyl optionally substitutedwith carboxyl, hydroxy or C₁₋₆ alkoxy, amido, (lower alkyl)amide, oramino optionally mono- or di-substituted with C₁₋₆ alkyl; (ii) C₃₋₇cycloalkyl or C₄₋₁₀ alkylcycloalkyl, both optionally substituted withhydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (lower alkyl)amide, oramino optionally mono- or di-substituted with C₁₋₆ alkyl; (iv) C₆ or C₁₀aryl or C₇₋₁₆ aralkyl, all optionally substituted with C₁₋₆ alkyl,hydroxy, amido, (lower alkyl)amide, or amino optionally substituted withC₁₋₆ alkyl; (v) Het or (lower alkyl)-Het, both optionally substitutedwith C₁₋₆ alkyl, hydroxy, amino optionally substituted with C₁₋₆ alkyl,amido, (lower alkyl)amide, or amino optionally substituted with C₁₋₆alkyl, or B is a carboxyl derivative of formula R₄—O—C(O)—, wherein R₄is (i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoyl,hydroxy, C₁₋₆ alkoxy, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido or (lower alkyl)amide; (ii) C₃₋₇ cycloalkyl, C₄₋₁₀alkylcycloalkyl, all optionally substituted with carboxyl, (C₁₋₆alkoxy)carbonyl, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido or (lower alkyl)amide; (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyloptionally substituted with C₁₋₆ alkyl, hydroxy, amido, (loweralkyl)amido, or amino optionally mono- or di-substituted with C₁₋₆alkyl; or (v) Het or (lower alkyl)-Het, both optionally substituted withC₁₋₆ alkyl, hydroxy, amino optionally mono- or di-substituted with C₁₋₆alkyl, amido or (lower alkyl)amido, or B is an amide derivative offormula R₄—N(R₅)—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyl optionallysubstituted with carboxyl, C₁₋₆ alkanoyl, hydroxy, C₁₋₆ alkoxy, amido,(lower alkyl)amido, or amino optionally mono- or di-substituted withC₁₋₆ alkyl; (ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, alloptionally substituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido,(lower alkyl)amido, or amino optionally mono- or di-substituted withC₁₋₆ alkyl; (iii) amino optionally mono- or di-substituted with C₁₋₃alkyl; (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, all optionally substitutedwith C₁₋₆ alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionallysubstituted with C₁₋₆ alkyl; or (v) Het or (lower alkyl)-Het, bothoptionally substituted with C₁₋₆ alkyl, hydroxy, amino optionallysubstituted with C₁₋₆ alkyl, amido or (lower alkyl)amide; and R₅ is H ormethyl, or B is thioamide derivative of formula R₄—NH—C(S)—; wherein R₄is (i) C₁₋₁₀ alkyl optionally substituted with carboxyl, C₁₋₆ alkanoylor C₁₋₆ alkoxy; (ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, alloptionally substituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amino oramido; Y is H or methyl; R³ is C₁₋₈ alkyl, C₃₋₇ cycloalkyl, or C₄₋₁₀alkylcycloalkyl, all optionally substituted with hydroxy, C₁₋₆ alkoxy,C₁₋₆ thioalkyl, acetamido, C₆ or C₁₀ aryl, or C₇₋₁₆ aralkyl; R² is S—R₂₀or O—R₂₀ wherein R₂₀ is pyridinyl, quinolyl, —CH₂-pyridinyl or—CH₂-quinolyl, all optionally mono-, di- or tri-substituted with R₂₁,wherein R₂₁ is C₁₋₆alkyl; C₁₋₆ alkoxy; lower thioalkyl; amino or amidooptionally mono- or di-substituted with C₁₋₆ alkyl, C₆ or C₁₀ aryl,C₇₋₁₆ aralkyl, Het or (lower alkyl)-Het; NO₂; OH; halo; trifluoromethyl;carboxyl; C₆ or C₁₀ aryl, C₇₋₁₆ aralkyl, or Het, said aryl, aralkyl orHet being optionally substituted with R₂₂, wherein R₂₂ is C₁₋₆ alkyl;C₃₋₇ cycloalkyl; C₁₋₆ alkoxy; amino; mono- or di-(lower alkyl)amino;(lower alkyl)amide; sulfonylalkyl; NO₂; OH; halo; trifluoromethyl;carboxyl or Het; or R² is

or R² is quinolinoxy unsubstituted, mono- or di-substituted with R₂ asdefined above; and P1 is:

 wherein R¹ is H, C₁₋₃ alkyl, C₃₋₅ cycloalkyl, or C₂₋₄ alkenyloptionally substituted with halo, and said R¹ at carbon 2 is orientatedsyn to the carbonyl at position 1, represented by the radical:

wherein at least one “Het” group is present in the compound of formula(I) and “Het” is as defined in claim 1; or a pharmaceutically acceptablesalt or ester thereof.
 43. A compound of formula I according to claim42, wherein B is a C₆ or C₁₀ aryl optionally substituted with C₁₋₆alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl, hydroxy, hydroxyalkyl, halo,haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amide, or aminooptionally mono- or di-substituted with C₁₋₆ alkyl; or B is Hetoptionally substituted with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkanoyl,hydroxy, halo, amido, (lower alkyl)amide, or amino optionally mono- ordi-substituted with C₁₋₆ alkyl; or B is R₄—SO₂ wherein R₄ is C₆ or C₁₀aryl, a C₇₋₁₄ aralkyl or Het all optionally substituted with C₁₋₆ alkyl;amido, (lower alkyl)amide; or B is an acyl derivative of formulaR₄—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyl optionally substituted withcarboxyl, hydroxy or C₁₋₆ alkoxy; or (ii) C₃₋₇ cycloalkyl or C₄₋₁₀alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl,(C₁₋₆ alkoxy)carbonyl; or (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl, alloptionally substituted with C₁₋₆ alkyl, hydroxy; or (v) Het optionallysubstituted with C₁₋₆ alkyl, hydroxy, amido or amino; or B is a carboxylderivative of formula R₄—O—C(O)—, wherein R₄ is (i) C₁₋₁₀ alkyloptionally substituted with carboxyl, C₁₋₆ alkanoyl, hydroxy, C₁₋₆alkoxy or amido, (lower alkyl)amide, amino optionally mono- ordi-substituted with C₁₋₆ alkyl; (ii) C₃₋₇ cycloalkyl, C₄₋₁₀alkylcycloalkyl, all optionally substituted with carboxyl, (C₁₋₆alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- ordi-substituted with C₁₋₆ alkyl; or (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl,all optionally substituted with C₁₋₆ alkyl, hydroxy, amino optionallysubstituted with C₁₋₆ alkyl; or (v) Het or (lower alkyl)-Het, bothoptionally substituted with C₁₋₆ alkyl, hydroxy, amido, or aminooptionally mono-substituted with C₁₋₆ alkyl; or B is an amide derivativeof formula R₄—N(R₅)—C(O)— wherein R₄ is (i) C₁₋₁₀ alkyl optionallysubstituted with carboxyl, C₁₋₆ alkanoyl, hydroxy, C₁₋₆ alkoxy, amido,(lower alkyl)amide, amino optionally mono- or di-substituted with C₁₋₆alkyl; (ii) C₃₋₇ cycloalkyl or C₄₋₁₀ alkylcycloalkyl, all optionallysubstituted with carboxyl, (C₁₋₆ alkoxy)carbonyl, amido, (loweralkyl)amide, amino optionally mono- or di-substituted with C₁₋₆ alkyl;and R₅ is H or methyl; or R₄ is (iii) amino optionally mono- ordi-substituted with C₁₋₃ alkyl; or (iv) C₆ or C₁₀ aryl or C₇₋₁₆ aralkyl,all optionally substituted with C₁₋₆ alkyl, hydroxy, amino or amidooptionally substituted with C₁₋₆ alkyl; or (v) Het optionallysubstituted with C₁₋₆ alkyl, hydroxy, amino or amido; or B is athioamide derivative of formula R₄—NH—C(S)—; wherein R₄ is: (i) C₁₋₁₀alkyl; or (ii) C₃₋₇ cycloalkyl; or Y is H; R³ is the side chain of Tbg,IIe, Val, Chg or:

R² is quinolinoxy unsubstituted, mono- or di-substituted with R₂₁ asdefined in claim 42, or R² is:

wherein R_(21A) is C₁₋₆ alkyl; C₁₋₆ alkoxy; C₆, C₁₀ aryl or Het; lowerthioalkyl; halo; amino optionally mono-substituted with C₁₋₆ alkyl; orC₆, C₁₀ aryl, C₇₋₁₆ aralkyl or Het, optionally substituted with R₂₂wherein R₂₂ is C₁₋₆ alkyl, C₁₋₆ alkoxy, amido, (lower alkyl)amide, aminooptionally mono- or di-substituted with C₁₋₆ alkyl, or Het; R_(21B) isC₁₋₆ alkyl, C₁₋₆ alkoxy, amino, di(lower alkyl)amino, (loweralkyl)amide, NO₂, OH, halo, trifluoromethyl, or carboxyl; P1 is:

and R¹ is ethyl, vinyl, cyclopropyl, 1 or 2-bromoethyl or 1 or2-bromovinyl.
 44. A compound according to claim 42 represented by theformula:

wherein B, R³, R², R¹ are as defined below: R¹ syn to Table 3 car- Cpd #B R³ R² boxyl 329 Boc t-Bu

vinyl


45. A compound according to claim 42 represented by the formula:

wherein R³, R_(21A) and R_(21B) are as defined below: Table 6 Cpd # R³R_(21A) R_(21B) 609 t-Bu

— ; 612 t-Bu

— ; 617 t-Bu

— ; and 620 t-Bu

— .


46. A compound according to claim 45, selected from the group consistingof compound #612, 617, and
 620. 47. A compound according to claim 42represented by the formula:

wherein R³ and R_(21A) are as defined below: Table 7 Cmpd # R³ R_(21A)705 t-Bu

; 715 t-Bu

; 716 t-Bu

; 721 t-Bu

; 724 t-Bu

; 725 t-Bu

; 727 t-Bu

; and 729 t-Bu

;


48. A compound according to claim 47, which is compound #715.
 49. Apharmaceutical composition comprising an anti-hepatitis C virallyeffective amount of a compound of formula I according to claim 1, or atherapeutically acceptable salt or ester thereof, in admixture with apharmaceutically acceptable carrier medium or auxiliary agent.
 50. Amethod of treating a hepatitis C viral infection in a mammal byadministering to the mammal an anti-hepatitis C virally effective amountof the compound of formula I according to claim 1, or a therapeuticallyacceptable salt or ester thereof.
 51. A method of treating a hepatitis Cviral infection in a mammal by administering to the mammal ananti-hepatitis C virally effective amount of the composition accordingto claim
 49. 52. A method of inhibiting the replication of hepatitis Cvirus by exposing the virus to a hepatitis C viral NS3 proteaseinhibiting amount of the compound of formula I according to claim 1, ora therapeutically acceptable salt or ester thereof.
 53. A method oftreating a hepatitis C viral infection in a mammal by administeringthereto an anti-hepatitis C virally effective amount of a combination ofthe compound of formula I according to claim 1, or a therapeuticallyacceptable salt or ester thereof with another anti-HCV agent.
 54. Amethod according to claim 53, wherein said other anti-HCV agent isselected from the group consisting of: α-or β-interferon, ribavirin andamantadine.
 55. A method according to claim 53, wherein said otheranti-HCV agent comprises an inhibitor of other targets in the HCV lifecycle, selected from: helicase, polymerase, metalloprotease or IRES. 56.A process for the preparation of a peptide analog of formula (I)according to claim 1 wherein P1 is a substituted aminocyclopropylcarboxylic acid residue, comprising the step of: coupling a peptideselected from the group consisting of: APG-P3-P2; or APG-P2; with a P1intermediate of formula:

wherein R¹ is C₁₋₆ alkyl, cycloalkyl or C₂₋₆ alkenyl, all optionallysubstituted with halogen, CPG is a carboxyl protecting group and APG isan amino protecting group and P3 and P2 are as defined above.
 57. Aprocess for the preparation of: a peptide analog of formula (I)according to claim 1, this process comprising the step of: coupling asuitably protected amino acid, peptide or peptide fragment with a P1intermediate of formula:

wherein R¹ is C₁₋₆ alkyl, cycloalkyl or C₂₋₆ alkenyl, all optionallysubstituted with halogen, and CPG is a carboxyl protecting group.
 58. Aprocess for the preparation of: a peptide analog of formula (I)according to claim 1, this process comprising the step of: coupling asuitably protected amino acid, peptide or peptide fragment with a P1intermediate of formula:

wherein CPG is a carboxyl protecting group.
 59. The process according toclaim 56, 57 or 58 wherein said carboxyl protecting group (CPG) isselected from the group consisting of: alkyl esters, aralkyl esters, andesters being cleavable by mild base treatment or mild reductive means.60. Method of preparing a composition for treating a hepatitis C viralinfection in a mammal comprising combining an anti-hepatitis C virallyeffective amount of the compound of formula I according to claim 1, or atherapeutically acceptable salt or ester thereof, with apharmaceutically acceptable carrier medium or auxiliary agent. 61.Method of preparing a composition for inhibiting the replication ofhepatitis C virus comprising combining a hepatitis C viral NS3 proteaseinhibiting amount of the compound of formula I according to claim 1, ora therapeutically acceptable salt or ester thereof, with apharmaceutically acceptable carrier medium or auxiliary agent. 62.Method of preparing a composition for treating a hepatitis C viralinfection in a mammal comprising combining an anti-hepatitis C virallyeffective amount of a combination of the compound of formula I accordingto claim 1, or a therapeutically acceptable salt or ester thereof, andan interferon with a pharmaceutically acceptable carrier medium orauxiliary agent.
 63. A compound of formula (I) according to claim 1,wherein each Het group is independently selected from the groupconsisting of diazepine, piperidine, 1,4-dioxane, 4-morpholine,pyridine, pyrimidine, or quinoline.